Cyclic peptide and a medicament, external preparation and cosmetic comprising said cyclic peptide

ABSTRACT

The present invention is aimed for providing a novel peptide with a high drug efficacy and strong effect, a medicament or external preparation comprising it, specifically a prophylactic or therapeutic for dermatitis, rhinitis or alopecia, or a hair growth stimulant, a hair growing agent, an antipruritic or a skin-care product. The present invention achieved said aim by providing a cyclic peptide having an amino acid sequence expressed by the Formula I or a derivative thereof or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence does not have a peptide bond that is not between the amino acids constituting the amino acid sequence.

RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No. 15/122,753, which is a national stage filing under 35 U.S.C. § 371 of International Application No. PCT/JP2016/065839, filed May 27, 2016, the entire contents of each of which are incorporated herein by reference in their entireties.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 22, 2020, is named I054170002US01-SUBSEQ-JRV and is 31 kilobytes in size.

TECHNICAL FIELD

The present invention relates to a cyclic peptide and a medicament, external preparation and cosmetic comprising said cyclic peptide.

BACKGROUND ARTS

BNP (brain natriuretic peptide) is a hormone which is synthesized and secreted in heart (predominantly in ventricles). BNP was isolated from and identified in pig brain in 1988, and it has been known since that BNP is secreted from ventricular myocardium of human, etc. An increase in cardiac stress or development of myocardial hypertrophy induces BNP secretion and increased in blood concentration. In general, BNP has activities such as diuretic activity, vasodilation, renin-aldosterone secretion suppressing action, sympatholytic activity, hypertrophy suppressing action. BNP is considered to a hormone which acts to protect the myocardium against damages caused by cardiac stress.

Amino acids constituting BNP differ slightly from species to species who produce BNP. Nevertheless it been revealed that its structure possesses a cyclic part and a tail part as a common structure (Non-Patent Literatures 1-2). For instance, wild-type human BNP has been known to consist 32 amino acid residues, and its fragments and derivatives are further proposed (Patent Literatures 1-6).

In Japan, BNP is currently not used as a therapeutic, but widely used in clinical practice as a biochemical marker for cardiac failure. In United States, however, it is used as a drug for alleviating symptoms of cardiac failure (trade name: Natrecor®). Recently, it has been proposed to utilize an external preparation comprising BNP for treating dermatitis, rhinitis, alopecia, etc., and also as a skin-improving agent (Patent Literature7-9).

PRIOR ART REFERENCES Patent Literature

-   [Patent Literature1] JP A 2007-525213 -   [Patent Literature2] JP A 2008-509746 -   [Patent Literature3] WO 2008/032450 -   [Patent Literature4] U.S. Pat. No. 6,028,055 -   [Patent Literature5] U.S. Pat. No. 5,114,923 -   [Patent Literature6] U.S. Pat. No. 6,818,619 -   [Patent Literature7] WO 2011/010732 -   [Patent Literature8] WO 2011/024973 -   [Patent Literature9] WO 2012/099258

Non-Patent Literature

-   [Non-Patent Literature] N. Akizukia, K. Kangawaa, N. Minaminob, H.     Matsou, FEBS Letters 1991 280 (2)357-362. -   [Non-Patent Literature2] Takei Y, Fukuzawa A, Itahara Y, Watanabe T     X, Yoshizawa Kumagaye K, Nakajima K, Yasuda A, Smith M P, Duff D W,     Olson K R. FEBS Lett. 1997 Sep. 8; 414 (2)377-80.

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

As described above, BNP is considered to be a useful substance for the therapy of various diseases. However, in order to obtain a better therapeutic effect there is a need for developing a substance with a higher drug efficacy and stronger effect and a faster- and longer-acting substance.

An improvement in the duration and fast-acting properties of the drug efficacy and effect is quite meaningful in light of reducing required number/frequency of treatment, relieving the sufferings by the patient/user, and improving the quality of life (QOL) and the mental, physical, social or intellectual satisfaction in dairy living of the user. For example, dermatitis is often accompanied by symptoms such as an itch, infiltration or hot flush in the affected site; the sufferings by the patient will be reduced if these symptoms can quickly be alleviated. When an alopecia therapeutic, hair growing agent or hair growth stimulant is employed, the QOL of the user can be improved if the therapeutic effect, hair growth-stimulating or hair growing effect can be achieved immediately.

The inventors focused on these points and tried to search for a novel substance by reference to the structure of BNP.

Accordingly, the object of the present invention is to provide a novel peptide having a strong drug efficacy and effect, and a medicament or an external preparation comprising such peptide, specifically a prophylactic or therapeutic for dermatitis, rhinitis and alopecia, and a hair growth stimulant, a hair growing agent, an antipruritic, a cosmetic and a skin-care product, etc.

Means to Solve the Problems

It was previously considered that the tail part of the wild-type BNP peptide structure (which is composed of a cyclic part and tail part) plays at least certain important role in the binding and selectivity of BNP to its receptor. However, the inventors focused on the cyclic part of said structure, deleted the tail part, and made an intensive research on the resulting cyclic peptide. Accordingly we have obtained totally new findings including the effects of such cyclic peptide, its high drug efficacy, faster-acting and longer-lasting effect, and further continued the study and finally completed the invention.

Accordingly, the present invention relates to the followings:

[1] A cyclic peptide having an amino acid sequence expressed by Formula I:

wherein, X¹ denotes Gly, Val, Ala, Ser or Thr, X² denotes Arg, Gln or His, X³ denotes Lys or Arg, X⁴ denotes Met, Leu or Ile, X⁵ denotes Ile or Val, X⁶ denotes Ser or Gly, X⁷ denotes Ser or Ala, X⁸ denotes Ser, Gln, Val, Ala, Thr, Leu, Ile or Met, X⁹ denotes Ser, Val, Ala or Thr, X¹⁰ denotes Gly or Arg, X¹¹ denotes Leu, Met, Ile, Val or Ala, X¹² denotes Gly, Ser or Ala, and the line connecting two Cys denotes a disulfide bond, and wherein the amino acid sequence does not have a peptide bond that is not between the amino acids constituting the amino acid sequence, or a derivative thereof or a pharmaceutically acceptable salt thereof. [2] The cyclic peptide according to [1], wherein

X² denotes Arg,

X⁴ denotes Met,

X⁶ denotes Ser,

X⁷ denotes Ser, and

X¹⁰ denotes Gly,

or a derivative thereof or a pharmaceutically acceptable salt thereof. [3] The cyclic peptide according to [1], wherein the amino acid sequence is selected from the amino acid sequences expressed by Formula (I-a)-Formula (I-e):

wherein, the line connecting two Cys denotes a disulfide bond, or a derivative thereof or a pharmaceutically acceptable salt thereof. [4] The cyclic peptide according to any one of [1]-[3] or a derivative thereof or a pharmaceutically acceptable salt thereof, wherein the derivative is substituted by a substituent which is capable of replacing a hydrogen atom, hydroxyl group, carboxy group, amino group or imino group in the cyclic peptide. [5] The cyclic peptide or a derivative thereof or a pharmaceutically acceptable salt thereof, which is formed by deleting 1 to 4 amino acids in the cyclic peptide expressed by any one of the Formulae (I-a)-(I-e) according to [3], or by replacing them with or adding them other amino acids, and which has an equal function with the cyclic peptide expressed by each of said formulae. [6] An external preparation comprising one or more cyclic peptides according to any one of [1]-[5] and/or a derivative thereof and/or a pharmaceutically acceptable salt thereof. [7] The external preparation according to [6], wherein the external preparation is an ingredient for a dermatitis therapeutic, dermatitis prophylactic, antipruritic, antiphlogistic, epidermis regeneration accelerating agent, wound epithelialization-accelerating agent or skin-care product. [8] The external preparation according to [7], wherein the skin-care product is for moisturizing, and/or for preventing or improving rough skin, and/or for sebum/acne care, and/or for irritation alleviation/anti-inflammation, and/or for skin-lightening, and/or for anti-aging, and/or for preventing/alleviating ultraviolet lesion, and/or for slimming, and/or for skin-cleansing. [9] The external preparation according to [6], wherein the external preparation is a bath agent, a body-cleansing agent or a hair-cleansing agent. [10] The external preparation according to [6], wherein the external preparation is an alopecia therapeutic, an alopecia prophylactic, a hair growing agent and/or a hair growth stimulant. [11] The external preparation according to [6], wherein the external preparation is a rhinitis therapeutic and/or a rhinitis prophylactic. [12] The external preparation according to [6], wherein the external preparation is a cosmetic. [13] The external preparation according to any one of [6]4121, wherein the formulation is a solid, semi-solid, powder, liquid, spray, ointment, cream, emulsion, gel or patch formulation. [14] The external preparation according to any one of [6]4131, wherein the external preparation is used as a pharmaceutical product, a quasi-drug or a cosmetic product. [15] A use of the cyclic peptide according to any one of [1]-[4] and/or a derivative thereof and/or a pharmaceutically acceptable salt thereof for preparing the external preparation. [16] A method of using the external preparation comprising applying the external preparation according to any one of [6]-[14] to the skin and/or mucosa of a subject. [17] The method of using the external preparation according to [16], wherein the mucosa is labial, oral, nasal, ocular or vaginal mucosa. [18] The method of using the external preparation according to [16], wherein the method is a method of treating and/or preventing dermatitis, a method of alleviating or resolving itch, a method of treating eczematous or other erosion or ulcer, or a method for skin-care. [19] The method of using the external preparation according to [16], wherein the method is a method of treating and/or preventing alopecia, and/or a method of stimulating hair growth, and/or a method of growing hair. [20] The method of using the external preparation according to [16], wherein the method is a method of treating and/or preventing rhinitis. [21] A medicament comprising one or more cyclic peptides according to any one of [1]-[5] or a derivative thereof or a pharmaceutically acceptable salt thereof. [22] The medicament according to [21], wherein the medicament is a therapeutic for hypertension, unstable angina, acute myocardial infarction, edematous diseases, renal failure, cardiac failure, immune diseases, obesity or metabolic syndrome.

The cyclic peptide described herein (hereinafter also referred to as “BNP cyclic peptide”, “B ring” or “B ring-compound”) is, as described above, derived from wild-type BNP. The wild-type BNP encompasses BNP from human, as well as BNPs from monkeys, pigs, birds and rats. Therefore, the B ring-compound also encompasses the B ring-compound from human, as well as those from monkeys, pigs, birds and rats.

Effect by the Invention

According to the present invention, novel cyclic peptides and compositions comprising the same can be provided. Such compositions are applied to an external preparation for preventing or treating dermatitis, rhinitis or alopecia, and further applied to a hair growth stimulant, a hair growing agent or an antipruritic, each of which are provided as a medicament, a quasi-drug, a skin-care product, or a cosmetic product. When an “external preparation” is referred alone in the present invention, it means an agent that is applied to skin or mucosa, whose utility is not limited to a medicament, quasi-drug, skin-care product and cosmetic product.

In general, the external preparation of the present invention has a significantly higher efficacy against dermatitis as compared to a conventional steroid external preparation, as well as an excellent immediate effect such that the symptoms start to be improved within three minutes in general. Its effect is great and long-lasting, and the amelioration period is long.

Furthermore, the external preparation of the present invention can, upon being applied onto the subject's skin or mucosa suffering a dermatitis, quickly relieve or eliminate perceptible symptoms that are or can be caused by dermatitis such as pruritus, soreness (pain), hot sensation, tautness, infiltration and erythema, improving the symptoms of the dermatitis.

Furthermore, the external preparation of the present invention exerts a moisturizing effect on the applied site, while exerting an effect to improve skin texture where the corneum layer is present at the applied site. It can thus exerts effects to improve skin texture, improves dry or rough skin, softens and moisturizes skin, reduce and diminish wrinkles, and improve roughened lip.

The external preparation of the present invention also has an effect of stimulating hair growth or promoting hair growth and at the same time preventing hair loss at the applied site when being applied to a decalvant site or a site where hair growth is stimulated. In this case, stimulated hairs tend to become terminal hairs and not white hairs. These effects are exhibited relatively quick as compared to other active agents that have previously been used in the therapeutics for alopecia, e.g., BNP, and the obtained effects are more significant.

Yet the external preparation of the present invention has an immediate, large and long-lasting effect effect on rhinitis with a long amelioration period.

Besides, since the cyclic peptide of the invention is a peptide sharing a part of its structure with BNP which is a hormone inherent in body, it causes little concern about side effects. It is also considered to have a small influence on homodynamics as long as used in an appropriate amount or used externally onto skin, etc. It therefore can be administered for prolonged period to a patient in need thereof, e.g., a patient with chronic dermatitis. Moreover, the external preparation of the invention causes no irritation when being taken externally and can be applied safely to a patient with sensitive skin, as well as to a child or woman, on face or neck, etc.

The reason why the cyclic peptide of the invention has a superior drug efficacy and effect as compared with wild-type BNP is not yet clear. However, according to our in silico analysis, it is suggested that the cyclic peptide of the invention is more easily and strongly bound to NPR-A receptor (GC-A) as compared with wild-type BNP. It is thus predicted that the cyclic peptide would have an excellent drug efficacy and effect, an excellent immediate effect in particular. As described above, B ring-compound is derived not only from human, but there also are B rings from animals of other species such as monkeys, pigs, birds and rat, and it has been confirmed that they have similar structures. From the results of the structural analysis described hereinbelow, it is strongly speculated that all these B rings exert similar effects as those of the B ring-compound from human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1

A diagram showing the result of applying BNP cyclic peptide (A) or a gel formulation (B) on either left or right side of the face of a subject having large wrinkles on the face.

FIG. 2

A diagram showing the effect before and after the application of either B ring gel formulation or BNP gel formulation to a patient with female pattern alopecia and alopecia pityroides.

MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention is explained in detail based on its suitable embodiments.

1. The cyclic peptide, a derivative thereof and a pharmaceutically acceptable salt thereof Firstly, the cyclic peptide of the invention, a derivative thereof and a pharmaceutically acceptable salt thereof are explained.

The cyclic peptide of the present invention has an amino acid sequence expressed by Formula I:

wherein, X¹ denotes Gly, Val, Ala, Ser or Thr, X² denotes Arg, Gln or His, X³ denotes Lys or Arg, X⁴ denotes Met, Leu or Ile, X⁵ denotes Ile or Val, X⁶ denotes Ser or Gly, X⁷ denotes Ser or Ala, X⁸ denotes Ser, Gln, Val, Ala, Thr, Leu, Ile or Met, X⁹ denotes Ser, Val, Ala or Thr, X¹⁰ denotes Gly or Arg, X¹¹ denotes Leu, Met, Ile, Val or Ala, X¹² denotes Gly, Ser or Ala, the line connecting two Cys denotes a disulfide bond, and wherein the amino acid sequence does not have a peptide bond that is not between the amino acids constituting the amino acid sequence.

Such cyclic peptide is, similar to previously known BNPs, considered to bind to a receptor NPR-A (also known as GC-A) having a guanylate cyclase domain and promote the production of cyclic guanosine monophosphate (cGMP), and has activities such as, for example, diuretic action, vasodilation, renin-aldosterone secretion suppressing action, sympatholytic activity and hypertrophy suppressing action. It has a superior drug efficacy and effect, particularly an excellent immediate effect, as compared to BNP.

In addition, as described hereinbelow, the above cyclic peptide can be used as an ingredient of an external preparation for a dermatitis therapeutic/prophylactic, a rhinitis therapeutic/prophylactic, an alopecia therapeutic/prophylactic, a hair growing agent, a hair growth stimulant, an antipruritic, etc., or as an ingredient of a skin-care product, a quasi-drug or a cosmetic product. The above cyclic peptide can also be used as an alternative for BNP in an medicament utilizing the activity of BNP as above, e.g., in an medicament for hypertension, unstable angina, acute myocardial infarction, edematous diseases, renal failure, cardiac failure, immune diseases, obesity or metabolic syndrome.

In another embodiment of the present invention, in the cyclic peptide of the invention, X¹-X¹² in the Formula (I) may be defined as one or more selected from the group consisting of following (1)-(12):

(1) X¹ denotes Gly, Val, Ala, Ser or Thr, (2) X² denotes Arg, Gln or His, (3) X³ denotes Lys or Arg, (4) X⁴ denotes Met, Leu or Ile, (5) X⁵ denotes Ile or Val, (6) X⁶ denotes Ser or Gly, (7) X⁷ denotes Ser or Ala, (8) X⁸ denotes Ser, Gln, Val, Ala, Thr, Leu, Ile or Met, (9) X⁹ denotes Ser, Val, Ala or Thr, (10) X¹⁰ denotes Gly or Arg, (11) X¹¹ denotes Leu, Met, Ile, Val or Ala, and (12) X¹² denotes Gly, Ser or Ala.

A preferred cyclic peptide of the present invention is a cyclic peptide of Formula (I), wherein X² denotes Arg, X⁴ denotes Met, X⁶ denotes Ser, X⁷ denotes Ser, and/or X¹⁰ denotes Gly (SEQ ID NO: 2).

Furthermore, in another embodiment of the invention, in the cyclic peptide of the invention, the amino acid sequence expressed by Formula (I) may be selected from SEQ ID NOs: 3-8 and SEQ ID NOs: 16-75.

Furthermore, in the cyclic peptide of the invention, the amino acid sequence expressed by Formula (I) is preferably selected from the amino acid sequences expressed by the Formula (I-a)-Formula (I-e):

wherein, the line connecting two Cys denotes a disulfide bond.

Each of the amino acid sequences expressed by these Formulae (I-a)-(I-e) is the cyclic part of human BNP (Formula (I-a)), swine BNP (Formula (I-b)), rat BNP (Formula (I-c)), rabbit BNP (Formula (I-d)) and murine BNP (Formula (I-e)), respectively. Therefore, the cyclic peptide having such an amino acid sequence will exhibit the aforementioned effects with more certainty. Because swine BNP cyclic peptide is consistent with those of avian (SEQ ID NO: 8, Cys-Phe-Gly-Arg-Arg-Ile-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Met-Gly-Cys, wherein, 1st Cys and 17th Cys form a disulfide bond), bovine (SEQ ID NO: 9, Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, wherein, 1st Cys and 17th Cys form a disulfide bond), feline (SEQ ID NO: 10, Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, wherein, 1st Cys and 17th Cys form a disulfide bond), canine (SEQ ID NO: 11, Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, wherein, 1st Cys and 17th Cys form a disulfide bond), and ovine (SEQ ID NO: 12, Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys, wherein, 1st Cys and 17th Cys form a disulfide bond), BNP cyclic peptides derived from these organisms also have similar effects as the cyclic peptide of the present invention. Among those mentioned above, it is particularly preferred that the amino acid sequence expressed by Formula (I) is the amino acid sequence expressed by Formula (I-a). In another embodiment of the invention, the cyclic peptide may be expressed by SEQ ID NOs: 16-75.

The cyclic peptide of the present invention encompasses a derivatized form of said cyclic peptide. Such a derivative can be used as such, as an active substance, or also used as a prodrug.

A derivative of the present invention can be obtained by adding (modifying) a known substituent to certain group of an amino acid in the cyclic peptide such as, for example, a hydrogen atom, hydroxyl group, carboxy group, amino group or imino group, or replacing it with a known replaceable substituent. Modification include, but not limited to, chemical modifications such as, for example, glycosylation, acetylation, phosphorylation and lipidation, to an amino acid within the cyclic peptide.

The addition (modification) or replacement of an amino acid within the cyclic peptide can occur in one group, or can occur in more than one group at the same time. Any known substituents can be used as long as being capable of replacing above groups and it goes without saying that such substituents naturally include, for example, protecting groups such as BOC.

The cyclic peptide of the invention further includes a mutated form of said cyclic peptide. Namely, a mutant of the present invention can be obtained by deleting an amino acid in the cyclic peptide, replacing it with or adding it another amino acid. The number of amino acid to be deleted, replaced with other amino acids or added is 4 or less, more preferably 3 or less, even more preferably 2 or less, and particularly preferable 1 or less. It also goes without saying that the deletion, replacement or addition of amino acids in the cyclic peptide may occur concurrently and independently to each other.

An amino acid that is capable of being replaced with another amino acid can be, in the case of human BNP (Formula (I-a)), exemplified as follows, without being limited thereto. The third amino acid from left, Gly, may be replaced with either Val, Ala, Ser or Thr. The forth amino acid from left, Arg, may be replaced with either Gln or His. The fifth amino acid from left, Lys, may be replaced with Arg. The sixth amino acid from left, amino acid, Met, may be replaced with either Leu or Ile. The ninth amino acid from left, Ile, may be replaced with Val. The twelfth amino acid from left, Ser, may be replaced with either Gln, Val, Ala, Thr, Leu, Ile or Met. The thirteenth amino acid from left, Ser, may be replaced with either Val, Ala or Thr. The fourteenth amino acid from left, Gly, may be replaced with Arg. The fifteenth amino acid from left, Leu, may be replaced with either Met, Ile, Val or Ala. The sixteenth amino acid from left, Gly, may be replaced with either Ser or Ala. Table 32 summarizes examples of replaceable amino acids in the cyclic peptide of the invention, though the invention will not be limited thereto.

Cyclic peptides in which one amino acid has been replaced include such as, for example, SEQ ID NOs: 16-44, though the invention will not be limited thereto. Cyclic peptides in which two amino acids have been replaced include such as, for example, SEQ ID NO: 45-58, though the invention will not be limited thereto. Yet cyclic peptides in which three amino acids have been replaced include such as, for example, SEQ ID NOs: 59-70, though the invention will not be limited thereto. Furthermore, cyclic peptides in which four amino acids have been replaced include such as, for example, SEQ ID NOs: 71-75, though the invention will not be limited thereto. It also goes without saying that five or more amino acids can be replaced by appropriately combining the aforementioned amino acid.

Deletion of one to several amino acids can take place in similar manner to the replacement of the aforementioned amino acids with other amino acids.

The number of amino acid which are to be deleted or replaced with other amino acids or added may be determined such that the cyclic peptide will have 80% homology, preferably 90% homology to the cyclic peptide of the invention.

In addition, the present invention may be a mutant as described above, and a derivative thereof. As long as it retains the effect of the invention, any mutant or a derivative thereof is encompassed in the cyclic peptide according to the invention. In another embodiment, it has at least a BNP activity. For purpose of improving the activity of the cyclic peptide of the invention, prolonging the effect of the invention, and/or increasing storage stability of the cyclic peptide of the invention, the cyclic peptide of the invention or the amino acids constituting said peptide may be altered in an appropriate manner. For example, an amino acid in the cyclic peptide of the invention may be chemically modified, some amino acids constituting the cyclic peptide may be deleted or replaced with other amino acids, and/or new amino acids may be added.

For example, the C-terminal group —COOH of one Cys of the cyclic peptide may be replaced with —COOR¹, —CONHR¹ or —CONR¹², and/or the N-terminal group NH₂ of the other Cys of the cyclic peptide may be replaced with —NHC(O)R¹ or —N(C(O)R¹)₂. Here, each appearance of R¹ is independently a branched or straight hydrocarbon group or an alkylene glycol chain or sugar chain having 1 to 20 carbon atoms. The number of carbon atoms in R¹ is preferably 1 to 10, more preferably 1 to 5, yet more preferably 1 to 2.

Pharmaceutically acceptable salts include, without being particularly limited, as the cyclic peptide of the invention or a derivative, such as for example, a salt with an inorganic base, a salt formed with an organic base, a salt formed with an inorganic acid, a salt formed with an organic acid, a salt formed with a basic or acidic amino acid. Examples of suitable salt formed with inorganic base include such as, for example, an alkaline metal salt such as a sodium salt and potassium salt; an alkaline earth meal salt such as a calcium salt and magnesium salt; and an aluminum salt and ammonium salt. Examples of suitable salt formed with organic base include such as, for example, a salt with an alkyl amine such as trimethyl amine or triethyl amine; a salt formed with a heterocyclic amine such as pyridine and picoline; a salt formed with an alkanol amine such as ethanol amine, diethanol amine and triethanol amine; a salt formed with a cycloalkyl amine such as cyclohexyl amine and dicyclohexyl amine; a salt formed with an alkylene diamine derivative such as N,N′-dibenzylethylenediamine. Examples of suitable salt formed with inorganic acid include such as, for example, a salt formed with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid or phosphoric acid. Examples of suitable salt formed with organic acid include such as, for example, a salt formed with a monocarboxylic acid such as formic acid, acetic acid, trifluoroacetic acid and propionic acid; a salt formed with a polyvalent carboxylic acid such as fumaric acid, oxalic acid and maleic acid; a salt formed with a oxycarboxylic acid such as tartaric acid, citric acid, succinic acid and malic acid; a salt formed with a sulfonic acid such as methane sulfonic acid, benzene sulfonic acid and p-toluene sulfonic acid; and a salt formed with benzoic acid. Examples of suitable salt formed with a neutral amino acid include such as, for example, a salt formed with glycine, valine or leucine; examples of suitable salt formed with a basic amino acid include such as, for example, a salt formed with arginine, lysine or ornithine; and examples of suitable salt formed with an acidic amino acid include such as, for example, a salt formed with aspartic acid acid or glutamic acid.

Among those mentioned above, a cyclic peptide composed of the amino acid sequence expressed by Formula (I) or a pharmaceutically acceptable salt is preferred. Namely, it is preferred that the amino acid sequence expressed by Formula (I) is not replaced.

A method for producing the cyclic peptide of the present invention, a derivative and pharmaceutically acceptable salt thereof may employ, without being particularly limited, for example, any known chemical synthetic or genetic engineering methods.

When the amino acid sequence as above is chemically synthesized, it may be synthesized by any chemical synthetic method, or any known method for peptide synthesis, for example, or solid-phase or liquid-phase synthetic method. Moreover, any commercial synthesizer (e.g., SHIMADZU Corporation: PSSM-8) may be used for synthesis.

A disulfide bond can be formed in the amino acid sequence, for example, by DMSO oxidation method or iodine oxidation method without being particularly limited. In this case, an intramolecular disulfide bond can be formed by treating a free sulfhydryl group or a sulfhydryl group protected by a protecting group with either DMSO or iodine (I2) to result in said cyclic peptide.

A protecting group includes such as, for example, 4-methylbenzyl group (Bzl (4Me)), trityl group (Trt), tert-butyl group, N-(acetyl) aminomethyl group (Acm). Deprotection can be carried out through appropriate treatment corresponding to these protecting group, for example, for 4-methylbenzyl group by treating with a strong acid, and for N-(acetyl) aminomethyl group by treating with iodine.

Next, if necessary, the cyclic peptide is derivatized to result in a derivative. Derivatization can be carried out by known method. Alternatively, a derivative of the cyclic peptide can be produced at the time of peptide synthesis by introducing in advance a substituent into the amino acid constituting the cyclic peptide.

Then, if necessary, a pharmaceutically acceptable salt is produced by ion exchange of the cyclic peptide or derivative thereof. Ion exchange can be carried out, for example, by bringing into the contact the cyclic peptide or derivative thereof with a desired acid or base.

On the other hand, when the peptide is synthesized by genetic engineering, it can be synthesized by any known method such as, for example, methods described in Sambrook J. et al., Molecular Cloning, A Laboratory Manual (4th edition) (Cold Spring Harbor Laboratory Press (2012)). For instance, a DNA fragment coding for the amino acid sequence expressed by Formula (I) is prepared at first. Preparation of the DNA fragment can be performed, for example, if it is a case in which the DNA fragment codes for the amino acid sequence expressed by Formula (I-a), by amplifying the DNA fragment by PCR using a vector comprising a full-length human BNP gene as template and primers designed to synthesize a defined DNA region. Alternatively, a DNA fragment can be chemically synthesized.

Then, the amplified DNA fragment is ligated into an appropriate vector to obtain a recombinant vector for protein expression. Next, the vector for protein expression is allowed to be taken up by target cells and the transformed cells are selected. Finally, the protein produced by the cells (i.e., the protein composed of the amino acid sequence expressed by Formula (I)) is collected.

Formation of disulfide bond, derivatization and salt formation in the collected protein can be carried out as described above.

The identification of the compound of interest such as a cyclic peptide can be confirmed by known procedures such as, for example, reverse-phase HPLC or mass spectroscopy.

The presence or absence of BNP activity in the obtained compound can readily be confirmed by known means. For instance, it can be confirmed by examining cGMP producing activity in NPR-A receptor-expressing cells.

2. External Preparation

Secondly, the external preparation of the present invention is explained.

The external preparation of the present invention comprises one or more cyclic peptide and/or a derivative thereof and/or a pharmaceutically acceptable salt thereof as described above. If two or more cyclic peptides are used, the number of different cyclic peptide to be mixed is not limited, though 2 to 3 are preferred in terms of preparation cost and convenience. It includes SEQ ID NO: 19, 29, 33, 36, 39 and 43, for example, but the invention is not limited to these and these cyclic peptide can appropriately be combined as long as it does not interfere the effect of the invention.

2.1 Application

The external preparation of the present invention can be used for following uses, for example, without being particularly limited, and in each case exerts remarkable effect which had not previously been achieved. The external preparation of the present invention is not limited to any particular use, and can be used as a pharmaceutical product, a quasi-drug and/or a cosmetic product depending on its drug efficacy and effect.

Hereinbelow, the invention will be described in detail for each of its use.

(1) Therapeutic/Prophylactic for Dermatitis

The external preparation of the present invention can be a a therapeutic and/or prophylactic for dermatitis.

The external preparation of the present invention can, upon being applied onto the subject's skin or mucosa suffering dermatitis, quickly relieve or eliminate various perceptible symptoms and conditions that are or can be caused by dermatitis such as pruritus, soreness (pain), hot sensation, tautness, erythema, infiltration, papule, lichenification, crust, exudation or skin desiccation, improving the symptoms of dermatitis. In addition, the external preparation of the present invention does not cause any irritation at the site of application.

Furthermore, such action of the external preparation comprising the cyclic peptide of the invention is effected more quickly and for prolonged time period as compared to that of an external preparation comprising BNP having the tail part. Although reasons for these advantageous effects of the cyclic peptide over such BNP are not clear, the cyclic peptide of the invention is considered to have an advantageous structure for binding to its receptor (NPR-A receptor) over BNP, thereby enabling faster binding to the NPR-A receptor in the vicinity of the affected site and at the same time prolonging its binding time. Moreover, it is considered that its relatively quick binding to the NPR-A receptor near the affected site can prevents the cyclic peptide from diffusing out of the affected site via bloodstream, etc., enabling it to stay around the affected site for relatively long time.

The external preparation of the present invention does not only suppress or prevent inflammation in dermatitis but also acts to restore and/or retain the barrier function of skin. Here, skin's barrier function works for protecting skin against entry of stimuli and saprophytes from external environment or for retaining moisture. The external preparation restores the barrier function, and thus able to prevent the progress or exacerbation of inflammation. The barrier function of skin is greatly affected by the alignment state of corneocytes in the corneum, i.e., state of skin texture and moisturization. The external preparation of the present invention acts to improve skin texture and to moisturize skin. Therefore, its effects of restoring and maintaining skin barrier function are also obvious.

The external preparation of the present invention is also effective on skin symptoms commonly called acne, i.e., such as comedones, red papule, pustula, cysts/tuberosity.

Moreover, a quick relief or elimination of perceptible symptoms or conditions will reduce the burden of the subject (patient) and improve the QOL of the patient, while preventing the patient from being bothered by itch or soreness and from acting to damage the affected site, for example, touching or scratching. This effect of the external preparation of the present invention to relieve or eliminate perceptible symptoms or conditions is, in general, exhibited within 10 minutes, preferably within 5 minutes, more preferably within 3 minutes after application.

In addition, the external preparation of the present invention can treat dermatitis in which steroid dermatosis has been developed, or dermatitis that is intractable by other common drugs for dermatitis therapy such as steroid and tacrolimus, for example dermatitis for which a sufficient therapeutic effect cannot be achieved by these formulations, or dermatitis which is resistant to these formulations, or dermatitis for which the use of these formulations is not suitable or desirable. Conventional widely-used steroid external preparations have significant problems upon terminating application that the severity returns to the pre-application level and that a rebound phenomenon may occur and exacerbate the condition. The external preparation of the present invention has no such problems as a rebound phenomenon.

Dermatitis is in general a disease which causes inflammation in skin or mucosa. Dermatitis is normally accompanied with one or more symptoms selected from acute eczema symptoms such as erythema, infiltrative erythema, papule, vesicle, pustula, infiltration, incrustation and desquamation; chronic eczema symptoms such as lichenification and pigmentation; and scales, crust (scab) attachment, scratching, scratch scar, prurigo nodularis, erosion, edema, oozing and squamatization.

Dermatitis herein is not particularly limited as long as it is a disease which is accompanied with inflammation in skin or mucosa and includes such as, for example, eczema and atopic dermatitis such as chronic eczema, dyshidrotic eczema, infantile xerotic eczema; contact dermatitis such as allergic contact dermatitis and primary irritant contact dermatitis; seborrheic dermatitis; asteatotic dermatitis; autosensitization dermatitis; stasis dermatitis; urticaria such as allergic urticaria (e.g., alimentary urticaria and drug-induced urticaria) and nonallergic urticaria (e.g., physical urticaria, solar urticaria and cholinergic urticaria); insect bite; drug eruption; psoriasis such as plaque psoriasis, guttate psoriasis, erythrodermic psoriasis, pustular psoriasis and psoriasis arthropathica; prurigo such as chronic prurigo, acute prurigo, gestational prurigo and nodular prurigo; rosacea; rosacea-like dermatitis; cutaneous vasculitis such as cutaneous allergic vasculitis; cutaneous pruritus such as systemic cutaneous pruritus, localized cutaneous pruritus, senile cutaneous pruritus and gestational pruritus; solar dermatitis; erythrosis; nummular dermatitis; localized scratching dermatitis; perioral dermatitis; pompholyx; keratosis pilaris; lichen planus, dyshidrotic eczema, dyshidrosis, miliaria and acne vulgarisacne vulgaris. The external preparation of the present invention can be applied to the therapy and prophylaxis of any of these diseases.

The external preparation of the present invention exerts an excellent effect especially on eczema, atopic dermatitis, contact dermatitis, seborrheic dermatitis, insect bite, allergic or nonallergic urticaria and psoriasis, preferably on eczema, atopic dermatitis, contact dermatitis and insect bite, more preferably on eczema and atopic dermatitis. Therefore, the external preparation of the present invention can be used for the purpose of the therapy and prophylaxis of at least one or more dermatitis selected from above-mentioned group.

(2) Ingredients of Cosmetic or Skin-Care Product

The external preparation of the present invention may be an ingredient for a cosmetic or skin-care product.

The external preparation of the present invention will, upon being applied to skin and/or mucosa, provide moisture to the applied site and exert moisturizing effect, preventing skin from dryness and roughness. In cases where corneum is present at the site of application, it exerts an effect of improving skin texture there. It also provides and retains moderate elasticity and flexibility of skin and mucosa, softens skin, gives skin and mucosa resilience and firmness. Moreover, it improves wrinkles (including fine and large wrinkles) and flabbiness at the applied site, and further diminishes the appearance of dullness and spots. By diminishing dullness and spots away, it consequently provides a skin-lightening effect.

As a result, the external preparation of the present invention can be used for the purpose of maintaining or improving skin and/or mucosal condition, without being particularly limited, for example, for obtaining at least one effects selected from a group consisting of preventing or improving dry skin, skin roughness, sensitive skin, roughened lips and appearance of large and fine wrinkles, maintaining skin or mucosal condition, anti-aging, and skin-lightening, or obtaining for at least one effects selected from the above effects. Skin roughness herein includes miliaria, chilblain, cracked skin, chapped skin, acne, diaper rashes, festering, chafed inner thighs and razor burns.

Specific utilities of the external preparation of the present invention as an ingredient for the cosmetic or skin-care product includes, without being particularly limited, for example, cosmetics for make-up such as face lotion, emulsion, serum, cream, cold cream, gel, mask, pack, powder, hand soap, perfume, deodorant, as well as foundation, face powder, eye shadow, eyeliner, mascara, eyebrow, blush, makeup base, lip stick, lip cream and nail polish, and furthermore cosmetics for hair such as shampoo, rinse, conditioner, hair color, hair tonic, styling agent and perm chemical, body cleansers such as face wash, cleansing and body soap, skin-care products such as body powder, after-shave lotion and pre-shave lotion, and bath agents.

The above effects of the external preparation of the present invention are quickly exhibited, within 10 minutes in general, preferably within 5 minutes, more preferably within 3 minutes after application depending on the type of the effect.

The above effects of the external preparation of the present invention last for relatively long time. In general, the effect lasts for 4 hours or more, preferably, 8 hours or more, more preferably 24 hours or more after it started to effect depending on the type of the effect.

(3) Antipruritic

The external preparation of the present invention can be an antipruritic.

As mentioned above, the external preparation of the present invention is suitable for using as antipruritic because it quickly exerts an excellent antipruritic effect at the applied site when being applied to skin or mucosa.

The antipruritic effect of the external preparation of the present invention as mentioned above is exhibited within 10 minutes in general, preferably within 5 minutes, more preferably within 3 minutes after application.

(4) Alopecia Therapeutic, Alopecia Prophylactic, Hair Growing Agent and Hair Growth Stimulant

The external preparation of the present invention can also be an alopecia therapeutic, alopecia prophylactic, hair growing agent and/or hair growth stimulant.

When the external preparation of the present invention is applied to a site of hair loss or a site where hair growth is to be stimulated, it acts to prevent hair loss and promote hair growth stimulation and hair growing at the applied site. It has effects such as nourishing hair, promoting hair growth, and improving or preventing hair thinning at the applied site. In this case, hairs to be stimulated to grow tend to become terminal hairs or hairs which are not white hairs. These effects are exhibited in relatively early stage as compared to other active ingredients previously used in alopecia therapeutics e.g., BNP, and the effects obtained are significant.

The external preparation of the present invention also has an improving or preventing effect on dermatitis as mentioned above. Therefore, it can improve or prevent skin inflammation associated with alopecia. Such effects are particularly advantageous when alopecia has been exacerbated due to skin condition of the site of application (such as scalp).

Moreover, the external preparation of the present invention exerts moisturizing and skin texture-improving effects at the applied site when being applied to skin as mentioned above. It can remove and suppress dandruff and itching, while giving moisture to hair and scalp, improving and preventing dryness and keeping hair and scalp healthy. It also can improve seborrhea.

The external preparation of the present invention also acts to alleviate or eliminate the perceptible symptoms or conditions as mentioned above, thereby reducing the burden of the subject (patient) and improving QOL of the patient. Moreover, it can prevent the patient from being bothered by itch or soreness and acting to damage the affected site, for example, touching or scratching, thereby preventing exacerbation of skin condition which may induce alopecia.

When the external preparation of the present invention is used as alopecia therapeutic or prophylactic, applicable alopecia includes such as, for example, alopecia as listed below, without being particularly limited. The external preparation can be used for the purpose of treating or preventing one ore more of these alopecia.

(Acquired Alopecia)

(i) Alopecia without accompanying scarring or skin lesion (alopecia areata, male pattern alopecia, seborrheic alopecia, alopecia pityroides, female pattern alopecia, gestational alopecia, malignant alopecia, senile alopecia, alopecia totalis, alopecia areata multilocularis, ophiasis, drug-induced alopecia, cancer chemotherapy-induced alopecia and radiation exposure-induced alopecia, traumatic/mechanical alopecia, malnutrition/metabolic disorder associated alopecia, endocrine dysfunction associated alopecia and telogen effluvium (post partum alopecia, alopecia after high fever)). (ii) Alopecia observed on skin lesion or pathologic skin (infection-induced alopecia, tumor-induced alopecia, inflammation-induced alopecia) (iii) Scarring alopecia (skin infection-induced alopecia, alopecia induced by infiltration of inflammatory cells)

(Congenital Alopecia)

Diffuse alopecia, congenital atrichia, alopecia in hereditary syndromes, localized alopecia, phakomatosis, aplasia cutis, congenital alopecia triangularis.

Among those mentioned above, the external preparation of the present invention exerts an excellent effect particularly on acquired alopecia, preferably on alopecia without accompanying scarring or skin lesion, more preferably on alopecia areata, male pattern alopecia, seborrheic alopecia, alopecia pityroides, female pattern alopecia, gestational alopecia, malignant alopecia, senile alopecia, alopecia totalis, alopecia areata multilocularis, ophiasis, drug-induced alopecia, cancer chemotherapy-induced alopecia and radiation exposure-induced alopecia, traumatic/mechanical alopecia, malnutrition/metabolic disorder associated alopecia, endocrine dysfunction associated alopecia and telogen effluvium. Therefore, the external preparation of the present invention can be used for the purpose of treating or preventing at least one or more alopecia selected from the above-mentioned group.

(5) Rhinitis Therapeutic and/or Prophylactic

The external preparation of the present invention can also be a rhinitis therapeutic and/or prophylactic.

Rhinitis is a disease caused by mucosal inflammation in nasal cavity and/or paranasal cavity and induces main symptoms such as nasal obstruction, rhinorrhea, sudden recurrent sneezing, as well as symptoms such as pruritus. In the present invention, “rhinitis” does not only includes rhinitis in a narrow definition which are accompanied with mucosal inflammation in nasal cavity, but also includes sinusitis accompanied with mucosal inflammation in paranasal cavity.

The external preparation of the present invention can improve or prevent various symptoms associated with rhinitis such as nasal obstruction, rhinorrhea, sneezing and pruritus, when being applied to nasal cavity mucosa and/or paranasal cavity mucosa. Especially these effects are exhibited quickly and last for a prolonged time as compared to a rhinitis therapeutic comprising BNP having the tail part.

Such effects of the external preparation of the present invention on rhinitis are exhibited within 8 minutes in general, preferably within 5 minutes, more preferably within 3 minutes after its application.

Also the effect of the external preparation of the present invention on rhinitis as described above lasts for 4 hours or more in general, preferably for 8 hours or more, more preferably for 24 hours or more after it started to effect.

When the external preparation of the present invention is used as a rhinitis therapeutic and/or prophylactic, applicable rhinitis includes such as, without being particularly limited, for example, infectious rhinitis including acute rhinitis and chronic rhinitis; hypersensitive non-infectious rhinitis including combined (flower hypersensitivity) rhinitis, rhinitis with rhinorrhea, congestive rhinitis, edematous rhinitis and dry-nose type rhinitis; irritant-induced rhinitis including physical rhinitis, chemical rhinitis and radiation rhinitis; and atrophic rhinitis and idiopathic granulomatous rhinitis; and sinusitis such as acute sinusitis, chronic sinusitis (maxillary empyema), eosinophilic sinusitis and paranasal cavity mycosis, The external preparation can be used for the purpose of treating or preventing one ore more of the above.

Combined rhinitis includes, for example, allergic rhinitis including perennial allergic rhinitis and seasonal allergic rhinitis, and nonallergic rhinitis including vasomotor (essential) rhinitis and eosinophilic rhinitis.

Rhinitis with rhinorrhea includes, for example, gustatory rhinitis, cold air-induced rhinitis and senile rhinitis.

Congestive rhinitis includes, for example, drug-induced rhinitis, psychogenic rhinitis, gestational rhinitis, endocrine rhinitis and cold rhinitis.

Edematous rhinitis includes, for example, aspirin-hypersensitive rhinitis.

Among those mentioned above, the external preparation of the present invention exerts an excellent effect particularly on hypersensitive non-infectious rhinitis, irritant-induced rhinitis and sinusitis, preferably on hypersensitive non-infectious rhinitis and chronic sinusitis, more preferably on combined (flower hypersensitivity) rhinitis, rhinitis with rhinorrhea, congestive rhinitis, edematous rhinitis and dry-nose type rhinitis, chronic sinusitis. Therefore, the external preparation of the present invention can be used for the purpose of treating or preventing at least one or more rhinitis selected from the above-mentioned group.

In terms of symptoms, since the external preparation of the present invention acts as described above, it can be used for any of rhinitis with sneezing/rhinorrhea, rhinitis with nasal obstruction and rhinitis with both symptoms.

In addition, the external preparation of the present invention exhibits an effect of improving rhinitis which cannot be cured by a rhinitis therapeutic that has conventionally been used such as steroid drug.

(6) Other External Preparations

The external preparation of the present invention can also be used for an application other than those described above for the effect of the cyclic peptide as described above. In this case, the external preparation of the present invention can set an objective that is not the effects of the cyclic peptide as described above. In this case, the cyclic peptide of the invention, a derivative and/or a pharmaceutically acceptable salt thereof is used for the purpose of assisting the main effect of the external preparation or adding another effect to the main effect.

Such application include such as, without being particularly limited, for example, body powder, deodorant, depilation agent, soap, body shampoo, bath agent, hand soap, perfume, sunscreen, and antiinflammatory agent and antifungal agent.

2.2 Formulation

The external preparation of the present invention can exert the effect of its active ingredient, i.e., the cyclic peptide or a derivative and/or pharmaceutically acceptable salt thereof, certainly and quickly in the vicinity of the applied site by being locally applied to the site of the interest (e.g., affected site) on skin or mucosa.

Such external preparation can be, without being particularly limited, for example, an agent for integument, eye drop, ear drop, nasal drop, buccal agent or suppository. Among these, when the external preparation of the present invention is a dermatitis therapeutic, a dermatitis prophylactic, an antipruritic, a skin-care product, an alopecia therapeutic, an alopecia prophylactic, a hair growing agent or a hair growth stimulant, it is preferably an agent for integument. On the other hand, when the external preparation of the present invention is a rhinitis therapeutic and/or prophylactic, it is preferably a nasal drop. Furthermore, when it is a therapeutic/prophylactic of a corneal disease, it is preferably an eye drop.

When the external preparation of the present invention is an agent for integument, it can be, without being particularly limited, for example, an external solid formulation, an external liquid formulation, a spray formulation, an ointment, an emulsion, a cream, a gel formulation or a patch.

An external solid formulation is a solid formulation for applying or spraying onto such as skin. Such an external solid formulation includes, for example, a powder form external formulation.

An external liquid formulation is a liquid formulation for applying onto such as skin. Such an external liquid formulation includes, for example, a lotion and liniment.

A spray formulation is a formulation for spraying an active ingredient in a mist, powder, foam or paste form onto skin. Such spray formulation includes, for example, an external aerosol and a pump spray formulation.

An ointment is a semi-solid formulation which is applied to skin and comprises an active ingredient dissolved or dispersed in a base. The ointment can also be a lip cream for locally applying to lips, etc.

A cream is a semi-solid formulation which is applied to skin and emulsified as either oil-in-water or water-in-oil type.

A gel formulation is a gelled formulation which is applied to skin. The gel formulation includes, for example, aqueous gel formulation and oil-based gel formulation.

A patch is a formulation which is attached to skin. The patch includes, for example, a tape or plaster.

A nasal drop is a formulation which is administered to nasal cavity or nasal mucosa. The nasal drop includes, for example, nasal powder formulation and a nasal drop. Among these, nasal drop is preferred.

In any formulation described above, the external preparation of the present invention comprises the cyclic peptide of the present invention and/or a derivative thereof and/or a pharmaceutically acceptable salt thereof as described above.

When the formulation is an external liquid formulation, an ointment, a cream or a gel formulation, the external preparation of the present invention comprises the cyclic peptide and/or a derivative thereof and/or a pharmaceutically acceptable salt thereof at a concentration of, for example, 0.0001 to 1000000 μg/g, preferably 0.001 to 10000 μg/g, more preferably 0.01 to 1000 μg/g, yet more preferably 0.1 to 100 μg/g. In another embodiment, it may comprises the cyclic peptide at a concentration of 1 to 800 μg/g or 3 to 500 μg/g.

When the formulation is a spray formulation, the external preparation of the present invention comprises in the stock solution of the spray formulation the cyclic peptide and/or a derivative thereof and/or a pharmaceutically acceptable salt thereof at a concentration of, for example, 0.0001 to 1000000 μg/mL, preferably 0.001 to 10000 μg/mL, 0.01 to 1000 μg/mL, more preferably 0.1 to 100 μg/mL, yet more preferably 1 to 100 μg/mL. In another embodiment, it may comprises the cyclic peptide at a concentration of 1 to 800 μg/mL or 3 to 500 μg/mL.

When the formulation is a patch, the external preparation of the present invention comprises the cyclic peptide and/or a derivative thereof and/or a pharmaceutically acceptable salt thereof at a concentration of, for example, 0.0001 to 1000000 μg/mL, preferably 0.001 to 10000 μg/mL, more preferably 0.01 to 1000 μg/mL, yet more preferably 0.1 to 100 μg/mL, particularly preferably 1 to 100 μg/mL. In another embodiment, it may comprises the cyclic peptide at a concentration of 1 to 800 μg/mL or 3 to 500 μg/mL.

When the formulation is a nasal drop, especially a liquid nasal drop, the external preparation of the present invention comprises in the nasal drop solution (nasal drop) the cyclic peptide and/or a derivative thereof and/or a pharmaceutically acceptable salt thereof at a concentration of, for example, 0.0001 to 1000000 μg/mL, preferably 0.001 to 10000 μg/mL, more preferably 0.01 to 1000 μg/mL, yet more preferably 0.1 to 100 μg/mL, particularly preferably 1 to 100 μg/mL. In another embodiment, it may comprises the cyclic peptide at a concentration of 1 to 800 μg/mL or 3 to 500 μg/mL.

The external preparation of the present invention can be formulated, for any formulation as described above, by using methods and constituent materials known to those skilled in the art.

Available constituent materials include such as, without being particularly limited, for example, a gelator, oily ingredient, higher alcohol, fatty acid, ultraviolet absorbing agent, ultraviolet scattering agent, powder, pigment, surfactant, polyhydric alcohol/sugar, polymer, bioactive ingredient, solvent, antioxidant, flavor and antiseptic agent.

Various organic or inorganic gelator compounds can be used.

An inorganic gelator compound includes such as, for example, hydrous or water-absorbable silicate, for example, aluminum silicate (e.g., bentonite), magnesium-aluminum silicate and colloidal silica.

As an organic gelator compound, a natural, semisynthetic or synthetic polymer can be used. natural and semisynthetic polymers include such as, for example, polysaccharides such as cellulose, starch, tragacanth, gum arabic, xanthane gum, agar, gelatin, alginic acid and a salt thereof (e.g., sodium alginate and a derivative thereof), lower alkylcellulose (e.g., methylcellulose or ethylcellulose), carboxy- or hydroxy-lower alkylcellulose (e.g., carboxymethylcellulose or hydroxypropylcellulose). A synthetic polymer includes such as, for example, carboxylvinyl polymer, sodium polyacrylate, (vinylmethyl ether/ethyl maleate) copolymer, polymethacrylate, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylate or polymethacrylate. Alternatively, as a gel formulation, commercially available gelators such as, for example, Lubrajel® NP, Lubrajel® CG, Lubrajel® DV, Lubrajel® MS, Lubrajel® OIL, Lubrajel® TW, Lubrajel® DS (Ashland Inc.) can also be used.

As an oily ingredient, for example, various ester, ether, hydrocarbon, silicone and fluorine oil phase ingredient, as well as animal and plant oils and a hardened oil thereof, and waxes of natural origin.

The ester oil phase ingredients include such as, for example, glyceryl tri(2-ethyl hexanoate), cetyl 2-ethylhexanoate, isopropyl myristate, butyl myristate, isopropyl palmitate, ethyl stearate, octyl palmitate, isocetyl isostearate, butyl stearate, butyl myristate, ethyl linoleate, isopropyl linoleate, ethyl oleate, isocetyl myristate, isostearyl myristate, isostearyl palmitate, octyldocecyl myristate, isocetyl isostearate, diethyl sebacate, diisopropyl adipate, isoalkyl neopentanoate, glyceryl tri(capryl-caprinate), trimethylol propane tri(2-ethylhexanoate), trimethylol propane triisostearate, pentaerythritol tetra(2-ethylhexaonate), cetyl caprylate, decyl laurate, hexyl laurate, decyl myristate, myristyl myristate, cetyl myristate, stearyl stearate, decyl oleate, cetyl ricinoleate, isocetyl myristate, isostearyl myristate, isocetyl palmitate, isostearyl palmitate, octyl stearate, isocetyl stearate, isodecyl oleate, octyldodecyl oleate, octyldodecyl linoleate, isopropyl isostearate, cetostearyl 2-ethylhexanoate, stearyl 2-ethylhexanoate, hexyl isostearate, ethylene glycol dioctanoate, ethylene glycol dioleate, propylene glycol dicaprinate, propylene glycol di(capryl-caprinate), propylene glycol dicaprylate, neopentyl glycol dicaprinate, neopentyl glycol dioctanoate, glyceryl tricaprylate, glyceryl triundecanoate, glyceryl triisopalmitate, glyceryl triisostearate, octyldodecyl neopentanoate, isostearyl octanoate, octyl isononanoate, hexyldecyl neodecanoate, octyldodecyl neodecanoate, isocetyl isostearate, isostearyl isostearate, octyldecyl isostearate, polyglycerol oleic acid ester, polyglycerol isostearic acid ester, dipropyl carbonate, dialkyl (C12-18) carbonate, triisocetyl citrate, triisoalkyl citrate, triisooctyl citrate, lauryl lactate, myristyl lactate, cetyl lactate, octyldecyl lactate, triethyl citrate, acetyltriethyl citrate, acetyltributyl citrate, trioctyl citrate, diisostearyl malate, 2-ethylhexyl hydroxystearate, di(2-ethylhexyl) succinate, diisobutyl adipate, diisopropyl sebacate, dioctyl sebacate, cholesteryl stearate, cholesteryl isostearate, cholesteryl hydroxystearate, cholesteryl oleate, dihydrocholesteryl oleate, phytosteryl isostearate, phytosteryl oleate, isocetyl 12-stearoylhydroxystearate, stearyl 12-stearoylhydroxystearate and isostearyl 12-stearoylhydroxystearate.

Hydrocarbon oil phase ingredients include such as, for example, squalane, liquid paraffin, α-olefin oligomer, isoparaffin, ceresin, paraffin, liquid isoparaffin, solid paraffin, polybutene, microcrystalline wax and vaseline.

Silicone oil phase ingredients include such as, for example, dimethylpolysiloxane, methylphenylpolysiloxane, methylcyclopolysiloxane, octamethylpolysiloxane, dacamethylpolysiloxane, dodecamethylcyclosiloxane, methyl hydrogen polysiloxane, polyether-denatured organo(polysiloxane), dimethylsiloxane-methylcetyloxysiloxane copolymer, dimethylsiloxane/methylstearoxysiloxane copolymer, alkyl-denatured organo(polysiloxane), terminal-denatured organo(polysiloxane), amino-denatured silicone oil, amino-denatured organo(polysiloxane), dimethiconol, silicone gel, acryl silicone, trimethyl siloxysilicate, silicone RTV gum.

Fluorine oil phase ingredients include such as, for example, perfluoropolyether, fluorine-denatured organo(polysiloxane), pitch fluoride, fluorocarbon, fluoroalcohol and fluoroalkyl-polyoxyalkylene co-denatured organo(polysiloxane).

Animal and plant oils and a hardened oil thereof, and waxes of natural origin include such as, for example, animal and plant oils and the hardened oil thereof such as beef tallow, hardened beef tallow, lard, hardened lard, horse oil, mink oil, orange roughy oil, fish oil, hardened fish oil, egg yolk, jojoba oil; plant oils and a hardened oil thereof such as avocado oil, almond oil, olive oil, cacao butter, apricot kernel oil, kukui nut oil, sesame oil, wheat germ oil, rice germ oil, rice bran oil, safflower oil, shea butter, soybean oil, evening primrose oil, camellia oil, corn oil, rapeseed oil, hardened rapeseed oil, palm kernel oil, hardened palm kernel oil, palm oil, hardened palm oil, peanut oil, hardened peanut oil, castor oil, hardened castor oil, sunflower oil, grape seed oil, jojoba oil, hardened jojoba oil, macadamia nut oil, meadowfoam oil, cottonseed oil, hardened cottonseed oil, palm oil, hardened palm oil, rose hip oil; waxes such as beeswax, beeswax having high acid value, lanolin, reduced lanolin, hardened lanolin, liquid lanolin, carnauba wax and montan wax.

Higher alcohols include such as, for example, lauryl alcohol, myristyl alcohol, cetyl alcohol, stearyl alcohol, isostearyl alcohol, oleyl alcohol, behenyl alcohol, 2-ethyl hexanol, hexadecyl alcohol, octyl dodecanol.

Fatty acids include such as, for example, caprylic acid, capric acid, undecylenic acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, isostearic acid, oleic acid, linoleic acid, linolenic acid, arachic acid, arachidonic acid, behenic acid, erucic acid, 2-ethyl hexanoic acid.

Ultraviolet absorbing agents include such as, for example, paraminobenzoate, amyl paraminobenzoate, ethyl dihydroxypropyl paraminobenzoate, glyceryl paraminobenzoate, ethyl paraminobenzoate, octyl paraminobenzoate, octyl dimethyl paraminobenzoate, ethylene glycol salicylate, octyl salicylate, triethanol aminesalicylate, phenyl salicylate, butyl phenyl salicylate, benzyl salicylate, menthyl salicylate, benzyl cinnamate, octyl paramethoxy cinnamate, 2-ethylhexyl paramethoxy cinnamate, glyceryl diparamethoxy cinnamate mono(2-ethylhexanoate), isopropyl paramethoxy cinnamate, paramethoxyhydrocinnamate diethanol amine salt, diisopropyl-disopropyl cinnamate ester mixture, urocanate, urocanate ethyl, hydroxymethoxy benzophenone, hydroxymethoxy benzophenone sulfonate and a salt thereof, dihydroxymethoxybenzophenone, sodium dihydroxymethoxybenzophenone sulfonate, dihydroxybenzophenone, dihydroxy dimethoxybenzophenone, hydroxyoctoxybenzophenone, tetrahydroxybenzophenone, butyl methoxy-dibenzoyl methan, 2,4,6-trianilino-p-(carbo-2-ethylhexyl-1-oxy)-1,3,5-triazine, 2-(2-hydroxy-5-methylphenyl) benzotriazole, methyl-O-aminobenzoate, 2-ethylhexyl-2-cyano-3,3-diphenyl acrylate, phenylbenzimidazole sulfate, 3-(4-methyl benzylidene) camphor, isopropyl dibenzoylmethan, 4-(3,4-dimethoxyphenylmethylene)-2,5-dioxo-1-imidazolidine propionate 2-ethylhexyl, and polymer derivatives or silane derivative thereof, titanium oxide and zinc oxide and dispersion thereof. The zinc oxide and titanium oxide may be surface-treated.

Dermal absorption auxiliary agents include such as, for example, acetic acid, sodium acetate, limonene, menthol, salicylic acid, hyaluronic acid, oleic acid, N,N-diethyl-m-toluamide (N,N-diethyl-3-methyl benzamide), n-butyl stearate, benzyl alcohol, isopropyl myristate, isopropyl palmitate, polypropylene glycol, crotamiton, diethyl sebacate, N-methylpyrrolidone, N-ethylpyrrolidone and lauryl alcohol.

Powders and pigments include such as, for example, pigments such as Pigment Red 104, Pigment Red 201, Pigment Yellow 4, Pigment Blue 1, Pigment Black 401, basic dyes, HC colors, disperse dyes, direct colors, lake pigments such as Pigment Yellow 4 AL lake, Pigment Yellow 203 BA lake; polymers such as nylon powder, silk powder, urethane powder, silicone powder, methyl polymethacrylate powder, cellulose powder, starch, silicone elastomer spherical powder and polyethylene powder; colored pigments such as yellow iron oxide, red iron oxide, black iron oxide, chromic oxide, carbon black, ultramarine, iron blue; white pigments such zinc oxide, titanium oxide, cerium oxide; extender pigment such as tare, mica, sericite, kaolin and tabular barium sulfate; pearl pigment such as titanium mica; metal salt such as barium sulfate, calcium carbonate, magnesium carbonate, aluminum silicate and magnesium silicate; inorganic powder such as silica, alumina; metal soap such as aluminum stearate, magnesium stearate, zinc palmitate, zinc myristate, magnesium myristate, zinc laurate, zinc undecylenate; bentonite, smectite, boron nitride, etc. The shape (spherical, rod-like, needle-like, tabular, amorphous, scaly, spindle-shaped) and particle diameter of these powders are not particularly limited.

These powders and pigments may be pre-treated by known conventional surface processing such as, for example, fluorine compound processing, silicone processing, silicone resin processing, pendant processing, processing with silane coupling agent, processing with titanium coupling agent, oil solution processing, N-acylated lysine processing, polyacrylic acid processing, metal soap processing, amino acid processing, lecithin processing, inorganic compound processing, plasma processing and mechanochemical processing.

As the surfactant, any of an anionic surfactant, a cationic surfactant, ampholytic surfactant and non-ionic surfactant can be used as appropriate.

Anionic surfactants include such as, for example, fatty acid soap, α-acylsulfonate, alkylsulfonate, alkylallylsulfonate, alkylnaphthalenesulfonate, alkylsulfate, POE alkylethersulfate, alkylamidesulfate, alkylphosphate, POE alkylphosphate, alkylamidephosphate, alkyloylalkyl taurine salt, N-acylamino acid salt, POE alkylether carboxylate, alkylsulfosuccinate, sodium alkylsulfoacetate, acylisethionate, acylated hydrolyzed collagen peptide salt and perfluoroalkyl phosphate ester.

Cationic surfactants include such as, for example, alkyltrimethylammonium chloride, stearyltrimethylammonium chloride, stearyltrimethylammonium bromide, cetostearyltrimethylammonium chloride, distearyldimethylammonium chloride, stearyldimethylbenzylammonium chloride, behenyltrimethylammonium bromide, benzalkonium chloride, propyldimethylhydroxypropylammonium behenic amide chloride, diethylaminoethyl-stearamide, dimethylaminopropyl-stearamide and lanolin derivative quaternary ammonium salt. Cationic surfactants also include tertiary amines such as fatty acid amide dialkyl amine and salts thereof.

Ampholytic surfactants include various ampholytic surfactants, for example, those of carboxybetain type, amidebetain type, sulfobetain type, hydroxysulfobetain type, amidesulfobetain type, phoshobetain type, aminocarboxylate type, imidazoline derivative type and amideamine type.

Non-ionic surfactants include such as, for example, propylene glycol fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, sorbitan fatty acid ester, POE sorbitan fatty acid ester, POE sorbitol fatty acid ester, POE glycerin fatty acid ester, POE alkyl ether, POE fatty acid ester, POE hardened castor oil, POE castor oil, POE-POP copolymer, POE-POP alkyl ether, polyether-denatured silicone alkanolamide laurate, alkylamine oxide, hydrogenated soybean phospholipid, hydrogenized soybean phospholipid, polymer surfactant and biosurfactant.

Natural surfactants may also be used, including such as, for example, lecithin, saponin and saccharide surfactant.

Polyhydric alcohols and sugars include such as, for example, ethylene glycol, diethylene glycol, polyethylene glycol, propylene glycol, dipropylene glycol, polypropylene glycol, glycerin, diglycerin, polyglycerin, 3-methyl-1,3-butanediol, 1,3-butylene glycol, sorbitol, mannitol, raffinose, erythritol, glucose, sucrose, fructose, xylitol, lactose, maltose, maltitol, trehalose, alkylated trehalose, mixed isomerized sugar, sulfated trehalose and pullulan. Chemically modificated forms of these can also be used.

Polymers include such as, for example, anionic polymer compounds such as acrylate ester/methacrylate ester copolymer, vinyl acetate/crotonate copolymer, vinyl acetate/crotonate/vinyl neodecanoate copolymer, methylvinyl ether maleate half ester, t-butyl acrylate/ethyl acrylate/methacrylate copolymer, vinylpyrrolidone/vinyl acetate/vinyl propionate copolymer, vinyl acetate/crotonatecopolymer (RUBISET CA: BASF), vinyl acetate/crotonate/vinylpyrrolidone copolymer, vinylpyrrolidone/acrylate copolymer, acrylate/acrylamide copolymer, vinyl acetate/butyl maleate/isoisobornyl acrylate copolymer, carboxyvinyl polymer, acrylate/methacrylate alkyl copolymer, and ampholytic acetate of dialkyl aminoethyl methacrylate polymer, ampholytic polymer compound such as octyl acrylamide acrylate/hydroxypropyl acrylate/butyl methacrylateaminoethyl copolymer, quaternized compound of vinylpyrrolidone/dimethylaminoethyl methacrylate, cationic polymer compounds such as methylvinyl imidazolium chloride/vinylpyrrolidone copolymer, non-ionic polymer compounds suchs as polyvinylpyrrolidone, vinylpyrrolidone/vinyl acetate copolymer, vinylpyrrolidone/dimethylaminoethyl methacrylate copolymer, vinylcaprolactam/vinylpyrrolidone/dimethylaminoethyl methacrylate copolymer. Polymer compounds of natural origin such as cellulose or derivatives thereof, keratin and collagen or derivatives thereof, calcium alginate, pullulan, agar, gelatin, tamarind seed polysaccharides, xanthan gum, carrageenan, high-methoxyl pectin, low methoxyl pectin, guar gum, gum arabic, crystalline cellulose, arabino galactan, can be karaya, gum tragacanth, alginates, albumin, casein, curdlan, gellan gum and dextran, and glucooligosaccharide, fucose containing polysaccharide, rhamnose containing polysaccharide can also suitably be combined.

Bioactive ingredients include substances which provide skin with some bioactivity when being applied to skin. Bioactive ingredients include those with such as, for example, skin-lightening, anti-inflammatory, anti-aging, ultraviolet protection, slimming, firming, antioxidation, hair growth stimulating/hair growing, suppressing hair growth, moisturizing, promoting circulation, antimicrobial/sterilization, cool/warm feeling, promoting wound cure, alleviating irritation, analgesic and cell-activating effects. Bioactive ingredients include such as plant extracts, seaweed extracts, vitamins and derivatives thereof, amino acids, various peptides other than the cyclic peptide, biopolymers such as sodium hyaluronate and mucopolysaccharide, intercellular lipid constituents such as ceramide, phytosphingosine, cholesterol and phytosterol, and analogues thereof, and enzymatic ingredients.

Examples of suitable combining ingredient include such as, for example, Angelica keiskei extract, avocado extract, sweet Hydrangea leaf extract, althea extract, arnica extract, aloe extract, apricot extract, apricot kernel extract, isoflavones, ginkgo extract, fennel extract, turmeric extract, oolong tea extract, Rose Fruit extract, echinacea extract, Baikal skullcup extract, Amur Corktree extract, Coptis japonica extract, barley extract, Hypericum erectum extract, Lamium album extract, cress extract, orange extract, cacao extract, desiccated sea water, seaweed extract, hydrolyzed elastin, hydrolyzed wheat powder, hydrolyzed silk, pumpkin seed extract, chamomile extract, carrot extract, Artemisia capillaris extract, licorice extract, hibiscus extract, Pyracantha fortuneana extract, kiwi extract, cinchona extract, cucumber extract, guanosine, gardenia extract, Sasa veitchii extract, Sophora flavescens extract, cranberry extract, walnut extract, grapefruit extract, clematis extract, chlorella extract, mulberry extract, gentian extract, tea extract, yeast extract, burdock extract, rice bran fermentation extract, rice germ oil, comfrey extract, collagen, cowberry extract, Asarum sieboldii Miq. extract, bupleurum extract, umbilical cord extract, salvia extract, soapwort extract, bamboo grass extract, crataegus extract, zanthoxylum extract, shiitake mushroom extract, Rehmannia root extract, Lithospermum root extract, Perilla frutescens extract, Tilia japonica (Miq.) extract, Filipendula multijuga extract, Paeonia lactillora extract, calamus extract, white birch extract, Equisetum arvense extract, Hedera helix extract, Crataegus oxyacantha extract, European elder extract, yarrow extract, peppermint extract, sage extract, tree mallow extract, cnidium extract, extract of Swertia japonica (Schult.) Makino, soybean extract, jujube extract, soybean fermentation extract, thyme extract, tea extract, clove extract, cogon extract, orange peel extract, Oenothera tetraptera extract, Centella asiatica extract, Terminalia sericea extract, dong quai extract, common marigold extract, peach kernel extract, bitter orange peel extract, extract of Houttuynia cordata Thunb., tomato extract, natto extract, ginseng extract, garlic extract, wild rose extract, hibiscus extract, Ophiopogon japonicus extract, parsley extract, honey, banana flower extract, hamamelis extract, parietaria extract, Isodon japonicus (Burm.) Hara extract, bisabolol, loquat extract, coltsfoot extract, butterbur flower extract, Poria Sclerotium extract, butcher's-broom extract, grape extract, propolis, luffa extract, safflower extract, peppermint extract, linden extract, tree peonyextract, hop extract, pine extract, horse chestnut extract, Lysichiton camtschatcense (L.) Schott extract, Sapindus mukurossi Gaertn. extract, Melissa extract, peach extract, bluebottle extract, eucalyptus extract, Saxifraga stolonifera extract, Citrus junos extract, coix seed extract, mugwort extract, lavender extract, apple extract, lytchee (Litchi) extract, lettuce extract, lemon extract, Chinese milk vetch extract, rose extract, rosemary extract, Roman chamomile extract and royal jelly extract.

Examples also include such as biopolymers such as deoxyribonucleic acid, mucopolysaccharides, sodium hyaluronate, sodium chondroitin sulfate, collagen, elastin, chitin, chitosan and hydrolyzed eggshell membrane; moisturizing ingredients such as amino acids, hydrolyzed peptides, sodium lactate, urea, sodium carbonate pyrrolidone, betain, whey, trimethyl glycine, polypeptides such as lysine/arginine condensate etc.; intercellular lipid constituents such as sphingolipid, ceramide, phytosphingosine, cholesterol, cholesterol derivative, phytosterol derivative, phospholipid and analogues thereof; anti-inflammatory agents such as ε-aminoaminocaproic acid, glycyrrhizic acid, β-glycyrrhetinic acid, lysozyme chloride, guaiazulen, hydrocortisone, tea tree oil, vitamins such as vitamin A and derivatives thereof, vitamin B2 and derivatives thereof, vitamin B6 and derivatives thereof, vitamin C and derivatives thereof, vitamin D and derivatives thereof, vitamin E and derivatives thereof, calcium pantothenate, biotin and nicotinic acid; active agents such as allantoin, diisopropylamine dichloroacetate, 4-aminomethylcyclohexane carbonate; antioxidants such as tocopherol, carotenoids, flavonoids, tannin, lignan and saponin; cell activating agents such as α-hydroxy acid and β-hydroxy acid; circulation accelerating agents such as γ-oryzanol and vitamin E derivative; wound curing agents such as retinol and retinol derivative; skin-lightening agents such as arbutin, kojic acid, placenta extract, sulfur, ellagic acid, linolenic acid, tranexamic acid, glutathione; hair growing agents such as cepharanthine, glycyrrhiza extract, capsicum tincture, hinokitiol, garlic extract iodide, pyridoxine hydrochloride, DL-α-tocopherol, DL-α-tocopherol acetate, nicotinic ac id, nicotinic acid derivative, calcium pantothenate, D-pantothenyl alcohol, acetyl pantothenyl ethyl ether, biotin, allantoin, isopropylmethylphenol, estradiol, ethinylestradiol, carpronium chloride, Benzalkonium chloride, diphenhydramine hydrochloride, Takanal, camphor, salicylic acid, vanillyl nonylate amide, vanillyl nonanoate amide, piroctone olamine, glyceryl pentadecanoate, L-menthol, mononitroguaiacol, resorcin, γ-aminobutyric acid, benzethonium chloride, mexiletine hydrochloride, auxin, female hormones, cantharides tincture, Cyclosporine, zinc pyrithione, hydrocortisone, minoxidil, polyoxyethylene sorbitan monostearate, peppermint oil, Sasanishiki extract.

Antioxidants include such as, for example, sodium bisulfite, sodium sulfite, erythorbic acid, sodium erythorbate, dilauryl thiodipropionate, tocopherol, tolyl biguanide, nordihydroguaiaretinoic acid, parahydroxyanisole, butyl hydroxyanisole, dibutyl hydroxytoluene, ascorbyl stearate, ascorbyl palmitate, octyl gallate, propyl gallate, carotenoids, flavonoids, tannin, lignans, saponins and plant extracts in which the antioxidative effect is recognized such as apple extract or clove extract.

Solvents include such as physiological saline, purified water, ethanol, lower alcohols, ethers, LPG, fluorocarbons, N-methylpyrrolidone, fluoroalcohols, volatile straight chain silicones and next-generation chlorofluorocarbons.

The cyclic peptide of the invention and/or a derivative thereof and/or a pharmaceutically acceptable salt thereof can be used for preparation of the external preparation as described above, and therefore the present invention also relates to a use of the cyclic peptide of the invention and/or a derivative thereof and/or a pharmaceutically acceptable salt thereof for preparing the external preparation.

3. Method of Using the External Preparation

Next, methods of using the external preparation of the invention is explained.

A method of using the external preparation of the invention comprises applying the external preparation of the present invention as described above to the skin and/or mucosa of a subject.

Subjects include human and vertebrates such as, without being particularly limited, for example, birds and mammals. Animals specifically include, for example, experimental animals including rodents such as mice, rats, gerbils, hamsters and guinea pigs, domestic animals such as pigs, goats, horses, sheep and minks, pet animals such as dogs and cats, primates such as human, monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans and chimpanzees. On the other hand, human may be excluded from the subjects.

Skin and/or mucosa of a subject to which the external preparation is to be applied may be skin or mucosa at any site of the subject, and the external preparation is applicable, for example, to skin or mucosa of head (scalp), face, neck, arms, torso, arms, hands or feet, etc.

Specific method for using the external preparation of the present invention is as follows.

(1) Method of Treating and Preventing Dermatitis

When the external preparation of the present invention is used as a therapeutic/prophylactic of dermatitis, the external preparation can be applied directly to the aimed site of skin and/or mucosa (e.g., the affected site where dermatitis has been developed).

The application frequency is, without being particularly limited, for example, 1 to 10 times a day, preferably 1 to 5 times a day, yet more preferably 1 to 3 times a day. Because the external preparation of the present invention has a long lasting time, it exerts a sufficient effect even if it is applied at relatively low frequency, for example, once a day.

In terms of dosage, the total amount of the cyclic peptide of the invention and a derivative thereof and a pharmaceutically acceptable salt thereof for one application can be, without being particularly limited, for example, 0.0001 to 1000000 μg/mL, preferably 0.001 to 10000 μg/mL, more preferably 0.01 to 1000 μg/mL, yet more preferably 0.1 to 100 μg/mL, particularly preferably 1 to 100 μg/mL. In another embodiment, each dose may comprise the cyclic peptide of the invention at a concentration of 1 to 800 μg/mL or 3 to 500 μg/mL.

(2) Method of Alleviating or Resolving Itch

When the external preparation of the present invention is used as an antipruritic, the external preparation can be applied directly to the aimed site of skin and/or mucosa.

The application frequency is, without being particularly limited, for example, 1 to 10 times a day, preferably 1 to 5 times a day, yet more preferably 1 to 3 times a day.

In terms of dosage, the total amount of the cyclic peptide of the invention and a derivative thereof and a pharmaceutically acceptable salt thereof for one application can be, without being particularly limited, for example, 0.0001 to 1000000 μg/mL, preferably 0.001 to 10000 μg/mL, more preferably 0.01 to 1000 μg/mL, yet more preferably 0.1 to 100 μg/mL, particularly preferably 1 to 100 μg/mL. In another embodiment, each dose may comprise the cyclic peptide of the invention at a concentration of 1 to 800 μg/mL or 3 to 500 μg/mL.

(3) Method of Using as a Cosmetic

When the external preparation of the present invention is used as a cosmetic, the external preparation can be applied directly to the aimed site of skin and/or mucosa.

The application frequency is, without being particularly limited, for example, 1 to 10 times a day, preferably 1 to 5 times a day, yet more preferably 1 to 3 times a day.

In terms of dosage, the total amount of the cyclic peptide of the invention and a derivative thereof and a pharmaceutically acceptable salt thereof for one application can be, without being particularly limited, for example, 0.0001 to 1000000 μg/mL, preferably 0.001 to 10000 μg/mL, more preferably 0.01 to 1000 μg/mL, yet more preferably 0.1 to 100 μg/mL, particularly preferably 1 to 100 μg/mL. In another embodiment, each dose may comprise the cyclic peptide of the invention at a concentration of 1 to 800 μg/mL or 3 to 500 μg/mL.

(4) Method of Treating and Preventing Alopecia, and Method of Stimulating Hair Growth and Method of Growing Hair

When the external preparation of the present invention is used as a therapeutic/prophylactic of alopecia, a hair growth stimulant or a hair growing agent, the external preparation can be applied directly to the aimed site (e.g., decalvant site of scalp or skin)

The application frequency is, without being particularly limited, for example, 1 to 10 times a day, preferably 1 to 5 times a day, yet more preferably 1 to 3 times a day.

In terms of dosage, the total amount of the cyclic peptide of the invention and a derivative thereof and a pharmaceutically acceptable salt thereof for one application can be, without being particularly limited, for example, 0.0001 to 1000000 μg/mL, preferably 0.001 to 10000 μg/mL, more preferably 0.01 to 1000 μg/mL, yet more preferably 0.1 to 100 μg/mL, particularly preferably 1 to 100 μg/mL. In another embodiment, each dose may comprise the cyclic peptide of the invention at a concentration of 1 to 800 μg/mL or 3 to 500 μg/mL.

(5) Method of Treating and Preventing Rhinitis

When the external preparation of the present invention is used as a therapeutic/prophylactic of rhinitis, the external preparation can be applied directly to the aimed site (e.g., nasal cavity mucosa).

The application frequency is, without being particularly limited, for example, 1 to 10 times a day, preferably 1 to 5 times a day, yet more preferably 1 to 3 times a day.

In terms of dosage, without being particularly limited, for example, for one application to each nasal cavity, the total amount of the cyclic peptide of the invention and a derivative thereof and a pharmaceutically acceptable salt thereof can be 0.0001 to 1000000 μg/mL, preferably 0.001 to 10000 μg/mL, more preferably 0.01 to 1000 μg/mL, yet more preferably 0.1 to 100 μg/mL, particularly preferably 1 to 100 μg/mL. In another embodiment, each dose may comprise the cyclic peptide of the invention at a concentration of 1 to 800 μg/mL or 3 to 500 μg/mL.

4. Medicament

Next, the medicament of the present invention is explained.

The medicament of the present invention comprises one or more cyclic peptides of the invention or derivatives thereof or pharmaceutically acceptable salts thereof.

As mentioned above, similarly to BNP, the cyclic peptide of the present invention or a derivative thereof or a pharmaceutically acceptable salt thereof binds to the receptor NPR-A (also known as GC-A), which has a guanylate cyclase domain, and promotes the production of cyclic guanosine monophosphate (cGMP). It is considered to have effects such as, for example, diuretic action, vasodilation, renin-aldosterone secretion suppressing action, sympatholytic activity and hypertrophy suppressing action. Also, the compounds of the invention is considered to have a superior efficacy and effect, particularly a superior immediate effect, as compared with BNP.

Therefore, the medicament of the present invention can be used for a similar object as a conventional medicament comprising BNP as an active agent.

Diseases for which the medicament of the present invention is applicable include such as, without being particularly limited, for example, the aforementioned various diseases as well as hypertension, unstable angina, acute myocardial infarction, edematous diseases, renal failure, cardiac failure, immune diseases, obesity and metabolic syndrome.

A formulation of the medicament of the present invention can be, without being particularly limited, addingly to the external preparations as mentioned above, for example, formulations for oral administration such as tablets, capsules, granules, powders, oral solution, syrup and oral jelly, formulations for oral application such as buccal tablets, buccal spray formulation, buccal semi-solid formulation and mouthwash, formulations for administration via injection such as an injection and infusion, dialysis formulation such as dialysis solution, as well as an inhalant, eye drop, ocular ointment, ear drop, vaginal tablet and vaginal suppository.

Working Examples

Hereinbelow, the present invention is further specifically illustrated by working examples. It should be noted that the present invention is not limited by these working examples.

1. Preparation of the Cyclic Peptide

Firstly, a cyclic peptide composed of the amino acid sequence expressed by Formula I-a is synthesized.

Specifically, a linear peptide consisting of 17 amino acids was formed by sequentially binding amino acids by solid-phase peptide synthesis using a peptide synthesizer. Subsequently, protecting groups at Cys1 and Cys17 were detached before treating with iodine (I2) to form a cysteine binding between oxidatively same amino acid residues, thereby forming a cyclic peptide.

A composition comprising the obtained cyclic peptide was purified by reverse-phase high performance liquid chromatography (reverse-phase HPLC) and then lyophilized to yield a purified cyclic peptide as white powder.

Mass spectroscopy was performed on the obtained cyclic peptide.

Conditions for HPLC are shown below:

Apparatus: Agilent 1100

Flow rate: 1.0 ml/min Eluent A: 0.1% trifluoroacetic acid/water Eluent B: 0.1% trifluoroacetic acid/acetonitrile Gradient: 80% Eluent B, isocratic

Conditions for Mass spectroscopy (MS) are shown below:

Apparatus: Thermo Finnigan LCQ Advantage

Ionization method: electrospray ionization Analytical method: ion trapping

The observed results, m/z=901.83 ([M+2H]²⁺), m/z=1801.84 ([M+H]⁺), confirmed that above peptide is in agreement with the theoretical values of the molecular weight (1802.07) and the mass number (1800.8069) calculated from the composition formula of the cyclic peptide of interest (C₇₂H₁₂₀N₂₄O₂₄S₃).

Furthermore, the purity of the above peptide was measured by HPLC using following conditions:

Column: Discovery C18, 4.6 mm×250 mm, particle diameter 5 micron

Column temperature: room temperature

Eluent A: 0.1% trifluoroacetic acid/water

Eluent B: 0.1% trifluoroacetic acid/acetonitrile

Gradient: 10 to 30% Eluent B/20 minutes

Flow rate: 1.2 ml/min

Temperature: room temperature

Injected volume: 20 μl

Detector: UV detector (detection wavelength: 215 nm)

The result of measurement confirmed that the purity of the obtained protein was 99.2%.

2. Preparation of Formulation 2.1 Preparation of Gel Formulation

Preparation of a gel-based formulation comprising a cyclic peptide composed of the amino acid sequence expressed by Formula I-a (in the working examples below, “B ring” is referred to mean a cyclic peptide expressed by such Formula I-a) (B ring gel formulation, Working Examples) and a gel-based formulation comprising human BNP (BNP gel formulation, Comparative Examples) were carried out as follows.

0.1 g of methyl-p-hydroxybenzoate (trade name: Mekkins-M, Ueno Fine Chemicals Industry, Ltd.), 0.2 g of phenoxyethanol and 3.0 g of 1,2-pentanediol were weighed into one same vessel, heated to 60 to 70° C. to make a homogenous solution, and this solution was poured into a mixer.

Next, added into the mixer 6.0 g of concentrated glycerin, and then the mixture of 0.44 g of carboxyvinyl polymer (trade name: Carbopol® 940, Lubrizol Advanced Materials Corporation) and 0.08 g of xanthane gum (trade name: KELTROL® T, CP Kelco, Inc.), and the mixuter was stirred with a paddle until they dispersed sufficiently.

Then 83.95 g of purified water was gradually added with stirring with a paddle. The mixer was heated to 70 to 80° C. while stirring with a paddle or disper until the dispersed contents were dissolved to give a solution. Subsequently, the disper was stopped and the solution was cooled immediately after confirming that the contents in the solution were dissolved. When the temperature of the mixer reached approximately 40° C., 6.0 g of Lubrajel® NP from Ashland Inc. (glycerin 2.7 g, carboxyvinyl polymer 0.06 g, sodium polyacrylate 0.018 g, water 3.222 g) was added to the solution, mixed uniformly with a paddle. Subsequently, 0.230 g of potassium hydroxide was further added to neutralize the solution, then the rotation of the paddle was stopped when the temperature of the mixer reached 25° C. to prepare a gel base.

Next, 20.1 mg of the the cyclic peptide (B ring) composed of the amino acid sequence expressed by Formula I-a was dissolved in 144 mL of physiological saline to obtain B ring solution. 0.131 mL of this solution was admixed with 10 g of the gel base obtained as described above, and the mixture was stirred uniformly to prepare a gel based formulation (B ring gel formulation) containing B ring at a concentration of about 1 μM (about 1.8 μg/g). Similarly, gel based formulations containing B ring at concentrations of about 0.3 μM (about 0.54 μg/g), about 0.5 μM (about 0.9 μg/g) and about 2.0 μM (about 3.6 μg/g) were prepared. In the working examples below, as long as it is specifically described otherwise, “B ring gel formulation” used contained B ring at a concentration of about 1 μM.

Next, 20.5 mg of human BNP-32 (American Peptide Company) was dissolved in 118 mL of physiological saline to obtain BNP solution. 0.2 mL of this solution was admixed with 10 g of the gel base obtained as described above, and the mixture was stirred uniformly to prepare a gel based formulation (BNP gel formulation) containing BNP-32 at a concentration of about 1 μM. Similarly, gel based formulations containing BNP-32 at concentrations of about 0.5 μM and 2.0 μM were prepared. In the working examples below, as long as being specifically described otherwise, “BNP gel formulation” used contained BNP at a concentration of about 1 μM.

2.2 Preparation of Nasal Drop

A nasal drop comprising the cyclic peptide (B ring) composed of the amino acid sequence expressed by Formula I-a (B ring nasal drop, Working Examples) and a nasal drop comprising human BNP (BNP nasal drop, Comparative Examples) were prepared as follows.

Firstly, the cyclic peptide (B ring) composed of the amino acid sequence expressed by Formula I-a was dissolved in physiological saline, the concentration was adjusted to prepare B ring nasal drop containing B ring at a concentration of about 1 μmol/1 (about 1.8 μg/g).

Next, B ring nasal drop was filled in a quantitative nasal spray container (AS ONE Corporation) and the amount to be sprayed for each administration was adjusted to 100 μl (0.1 ml), resulting B ring nasal drop.

Similarly, a BNP nasal drop comprising human BNP (American Peptide Company) at a concentration of 1 μmol/1 was obtained, filled in a quantitative nasal spray container (AS ONE Corporation), and the amount to be sprayed for each administration was adjusted to 100 μl (0.1 ml), resulting BNP nasal drop.

3. Confirming Therapeutic Effect on Dermatitis 3.1 Confirming the Effect of B Ring Gel Formulation

For subjects suffering various dermatitis, B ring gel formulation as described was applied onto the affected site, and changes in symptoms before and after the application were observed. When possible, as a comparative example, BNP gel formulation as described was applied onto another affected site on the same subject where B ring gel formulation had not been applied, and changes in symptoms before and after the application were observed. For pruritus, VAS (Visual Analogue Scale) was used for evaluating each affected site on 10 stages.

The examination results are shown in Tables 1 to 7 along with age, sexuality, symptoms of the subject, and formulation given to the subject.

TABLE 1 Pruritus before and after Pre-application symptoms Post-application symptoms application Case Treatment Disease (severity) (severity) (VAS) Notes D1 2 μM atopic Face: presented with Face: aches removed 2 min after 1 face 8→0 Age: 30's B ring gel dermatitis erythema, scales and skin application. Desiccation, scales and right arm 7→0 Sex: male formulation desiccation (moderate erythema

 After 3 days of 1 application disease). application, erythema, scales,

Limbs: presented with and desiccation remarkably remitted erythema, lichenification, (insignificant). skin desiccation and Right forearm: itches stopped to some scratch scar (severe). extent after 1.5 min on B ring-applied site. No itching or tingling after 2 min.

 remitteed after 4 min. Desiccation improved. Skin moisturized and soft, with natural appearance. No subjective discomfort (moderate). 2 μM Face:

 realized after 4 min. face 8→4 BNP gel erythema and scales more distinguished left arm 7→4 formulation than B ring side. 1 application Left forearm: after 4 min, skin was still dry. No improvement in lichenification. Rough and stiff skin (severe). D2 1 μM atopic Flushing, infiltrating erythema, Right back: itches stopped after 3 min. 8→0 Age: 20's B ring gel dermatitis scales, many scratch scars Flushing erythema, infiltration and Sex: male formulation erythroderma (severe) accompanied by scales remitted. Skin became soft 1 application strong itches causing sleep (moderate). 1 μM disruption and desire to Left back: still iching after 3 min. 8→4 BNP gel scratch. no remission of flushing erythema, formulation infiltration and scales (severe). 1 application D3 0.5 μM atopic Presented with infiltrating Right back: no tingling itches 30 sec 6→0 Age 10's B ring gel dermatitis erythema, scales, papules, after application. Skin felt normal. Sex: male formulation (moderate) accompanied by Erythema, infiltration and scales 1 application tingling itches. remitted after 4 min. (mild). 0.5 μM Left back: still itching after 4 min, 6→6 BNP gel with no remission of erythema, formulation infiltration and scales (moderate). 1 application

indicates data missing or illegible when filed

TABLE 2 Pruritus before and after Pre-application symptoms Post-application symptoms application Case Treatment Disease (severity) (severity) (VAS) Notes D4 2 μM atopic Presented with infiltrating Right back: itches relieved just after 10→0 Age: 20's B ring gel dermatitis erythema, scales, many application. No itches after 2 min with Sex: male formulation scratch scars with exudation remission of erythema and infiltration 1 application (severe) accompanied by (moderate). strong itches that cause sleep Forehead: itches reduced after 1.5 min. disruption on the back, After 5 min skin was soft with no lichenification, erythema, lichenification. Erythema and scales scales and scratch scars on remitted. Wound quickly healed. Scratch forehead (severe). scars epithelized. 2 μM Back: presented with many Left back: still itching after 4 min with no 10→3 BNP gel scratch scars with exudation, remission of erythema and infiltration formulation infiltration, erythema, (severe) 1 application crusts (severe). Forehead: no improvement in lichenification, desiccation and scales, with remaining itches (severe) D5 1 μM atopic Presented with infiltrating Right face: itches, edema and infiltration 9→0 Age: 20's B ring gel dermatitis erythema, edema and crusts remitted/relieved just after application. Sex: male formulation (severe) accompanied by Skin felt no itch and moisturized after 1 1 application strong tingling ithces. min (moderate). Back: presented with many No itches at all after 2 min. Edema and scratch scars with exudation, erythema remitted. Right eye became infiltration, erythema, crusts easy to open. 1 μM and papules (severe). Left face: still itching after 3 min with no 9→4 BNP gel remission of erythema, infiltration and formulation edema. Being difficult to open left eye 1 application (severe). D6 1 μM atopic Presented with lichenification, Right back: itches improved/relieved 6→0 Age: 10's B ring gel dermatitis

, erythema, after 3 min. Lichenification,

Sex: male formulation

scales, papules and many erythema, scales, scratch 1 application scratch scars (severe) scars all improved as compared to left. accompanied by strong itches. Skin was soft and moisturized Back: presented with many (moderate) 1 μM scratch scars with exudation, Left back: still itching after 4 min with no 6→3 BNP gel infiltration, erythema, crusts remission of erythema and infiltration formulation and papules (severe). (severe) 1 application Forehead: lichenification, desiccation and scales not improved, with itches left (severe).

indicates data missing or illegible when filed

TABLE 3 Pruritus before and after Pre-application symptoms Post-application symptoms application Case Treatment Disease (severity) (severity) (VAS) Notes D7 0.3 μM atopic Presented with infiltrating No itches after 1 min. Erythema 10→0 Intractable Age: 20's B ring gel dermatitis erythema, papules, exudation remitted and exudation stopped stroid- Sex: male formulation and many scratch scars with after 10 min. 1 application/day resistance 1 application crust (severe) accompanied for 3 days cleared of infiltrating by strong itches that cause erythema, papules, exudation and sleep disruption. scratch scars. Remarkably improved crusts, partly left mild erythema (mild). D8 0.5 μM atopic Presented with erythema, 5 days after application, erythema, 8→1 Intractable Sex: male B ring gel dermatitis infiltration, scales, infiltration, scales, crusts and steroid- formulation crusts, scratch scars and wound quickly cured with little resistance 1 application, skin desiccation (severe). scratch scars left (mild). 5 days Desiccation remarkably remitted Skin moisturized and soft. D9 B ring gel atopic Left forearm presented with Itches removed in 2 min (VAS 0). 10→0 Age: 30's formulation dermatitis many scratch scars with Erythema and exudation improved. Sex: male 2 applications/ exudation, infiltration, Severity of rash improved to day 1 day erythema, papules, moderate disease level after accompanied with tingling 5 min. After 1 more application strong itches (VAS10) given on the same night, (severe) there was no itches for 3 days thereafter. Scratch scars epithelized. D10 1 μM atopic Presented with infiltrating Right forearm: itches topped after 8→0 Age: 50's B ring gel dermatitis erythema, scales, papules and 3 min. Erythema and infiltration Sex: female formulation many scratch scars (severe) improved (moderate). 1 application accompanied by intermediate 1 μM strong itches Left forearm: still itching after 8→4 BNP gel 4 min with no remission of erythema formulation and infiltration (severe) 1 application D11 1 μM atopic Presented with infiltrating Right face: immediately after 10→0 Age: 40's B ring gel dermatitis erythema, scales and many application, itches and skin tautness Sex: female formulation scratch scars (severe) relieved, with erythema getting better. 1 application accompanied by drastic No itching after 1 min. Erythema, itches and tautness. infiltration and desiccation Back: presented with many remitted (moderate). 1 μM scratch scars with exudation, Left face: still itching after 3 min, 10→4 BNP gel infiltration, erythema, having erythema, infiltration and edema formulation crusts and papules (severe). with swollen feeling (severe). 1 application

TABLE 4 Pruritus before and after Pre-application symptoms Post-application symptoms application Case Treatment Disease (severity) (severity) (VAS) Notes D12 1 μm atopic Strong tingling itches, Right face: itches remitted after 3 min. 10 → 1 Age: 40's B ring gel dermititis disrupted sleep, infiltrating Edema, erythema and infiltration started Sex: formulation erythema, scales, exudation, to be remitted. After 30 min, anti- female 1 application cracks, crusts, scratch scars; inflammatory effect observed, exudation eyes cannot be opened due to stopped, wound epithelization promoted, exudate sealing eyelids cracks almost epithelized. Edema, (severe). erythema and infiltration remitted to some extent. Eyelids became openable steadily. 1 μm Left face: still having tingling itches after 10 → 6 BNP gel 3 min with no remission of erythema and formulation infiltration. Itches till left after 30 min. 1 application D13 1 μm atopic Obvious infiltration, erythema Right face: erythema started to be 8 → 0 Age: 20's B ring gel dermititis and papules all over the face, remitted and itches removed just after (immediate Sex: formulation accompanied with strong application. Erythema and infiltration after female 1 application itches and hot flash (severe). remitted after 4 min (moderate). application) 1 μm Left face: itching with hot flush and 8 → 0 BNP gel redness just after application. Itches (after 4 formulation removed after 4 min. minutes) 1 application D14 1 μm atopic Hand eczema of which main Right hand: itches removed after 3 min 9 → 0 Steroid- Age: 20's B ring gel dermititis symptom is contact dermititis (VAS0). Lichenification improved, skin resistance; Sex: formulation due to detergent and got soft, erythema and infiltration intractable female 1 application disinfectant. Presented with remitted (moderate). lichenification 1 μm lichenification, erythema, Left hand: no improvement in 9 → 4 which cannot BNP gel scales and scratch scars lichenification and erythema after 10 min be remitted formulation (severe). Sleep disruption by (severe). within this 1 application strong itches (VAS9). short time (3 min) by conventional therapy.

TABLE 5 Pruritus before and after Pre-application symptoms Post-application symptoms application Case Treatment Disease (severity) (severity) (VAS) Notes D15 1 μm atopic Presented with erythema, Erythema, infiltration, scales and crusts 9 → 1 Age: 20's B ring gel dermititis infiltration, scales, crusts, wound epithelization improved 3 days Sex: formulation scratch scars (severe), with after application. Scratch scars female 1 application/ strong itches. significantly improved. Only mild day erythema and scales observed (mild). 3 days Desiccation relieved, improved and moisturzed skin texture. Itches and scratch scars significantly improved. D16 1 μm contact Contact dermititis due to hair Right side: tingling itches removed after 8 → 0 Age: 60's B ring gel dermititis dye with erythena, edema 1 min. Itches eliminated after 3 min Sex: formulation and infiltration along hairline (VAS0), felt better than left. Erythema female 1 application (moderate disease) and infiltration remitted. accompanied by strong Anti-inflammatory effect observed. tingling itches (VAS8) Edema improved (mild). 1 μm Left side: symptoms less improved than 8 → 4 BNP gel right (moderate). formulation 1 application D17 1 μm chronic Presented with infiltrating Right face: itches stopped just after 10 → 0 Age: 40's B ring gel eczema erythema, scales, application. Erythema remitted. No Sex: formulation lichenification, crusts, papules scales and desiccation after 2 min. Skin female 1 application and skin desiccation (severe) was moisturized (moderate). accompanied by drastic itches Forehead: itches controlled after 1.5 min. and tautness. No lichenification after 5 min. Skin got soft. Erythema and scales remitted, scratch scars epithelized and wound quickly healed. 1 μm Left face: still itching and taut after 4 min 10 → 5 BNP gel with no remission of desiccation, scales, formulation erythema and infiltration (severe). 1 application D18 1 μm eczema Presented with erythema, After 15 min, erythema, edema, 8 → 0 Age: B ring gel edema, infiltration, scales and infiltration and scales significantly Sex: formulation papules (severe). remitted and skin texture improved female 1 application (mild). 1 μm After 15 min, still many scales, 8 → 4 BNP gel erythema, edema and infiltration left. formulation Skin texture is rough and still itching. 1 application

TABLE 6 Pruritus before and after Pre-application symptoms Post-application symptoms application Case Treatment Disease (severity) (severity) (VAS) Notes D20 1 μm wheal, Presented with multiple Right lower limb: itches removed just 10 → 0 Age: 20's B ring gel insect bites wheals, erythema and very after application. Erythema started to be Sex: formulation strong itches on both lower remitted after 1.5 min. Wheals female 1 application limbs. disappeared in about 1 hour. D31 1 μm wheal, Presented with multiple Left lower limb: itches removed 1 min 10 → 0 Age: 30's B ring gel insect bites wheals and very strong itches after application, wheals remitted after 2 Sex: formulation on right lower limb. min. female 1 application D21 1 μm childhood Presented with desiccation, Right back: itches stopped after 30 sec. 8 → 0 Age: 10's B ring gel xerotic many milary large papules, Desiccation, scales and erythema Sex: formulation eczema desquamation, strong pruritus, improved in a few minutes, Skin got female 2 applications/ and many scratch scars on moisturized. After 3 days, wounds day trunk; incrustation, erythema quickly epithelized leaving one scratch 3 days and lichenification due to scar. Skin is smooth and moisterized scratching (severe). with no desiccation (insignificant). 1 μm Left back: desiccation, scales and 8 → 3 BNP gel erythema left after few minutes. formulation 1 application D32 1 μm parapsoriasis Presented with multiple Scales, erythema and infiltration remitted N/A Age: 40's B ring gel guttata erythrokeratoderma with after 4 min. Sex: formulation sticking small scales on back. female 1 application D33 0.5 μm psoriasis Both lower limbs presented Scales and erythema started to be N/A Age: 60's B ring gel vulgaris with many red plaques with remitted 10 min after 1st application. Sex: male formulation attached small scales having After 1 week, no scales left. 2 applications/ clear margin. day 1 week D34 2 μm psoriasis Many red plaques with Back psoriasis: thick scales started to be N/A Age: 30's B ring gel vulgaris attached thick scales having remitted in 3 min after application, Sex: male formulation clear margin presented on almost disappeared after 30 min. 1 application back and neck. Neck psoriasis: erythema and scales remitted in 3 min after application.

TABLE 7 Pruritus before and after Pre-application symptoms Post-application symptoms application Case Treatment Disease (severity) (severity) (VAS) Notes D23 2 μm cyshidrotic Fingers presented with many Erythema and bullae remitted in 2 min N/A Intractable Age: 30's B ring gel eczema intractable bullae, being after application. After 1 day, erythema with Sex: male formulation dyshidrosis eczematous with erythema and scales remitted and bullae steroid 2 applications/ and cracks. disappeared. ointment. day 1 day D24 2 μm miliaria Red small papules and flare Pruritus, erythema and red small N/A Age: 50's B ring gel hyper- with itches. papules remitted after 3 min. Sex: male formulation hidrosis 1 application D25 1 μm chronic Many wheals with drastic Pruritus removed just after application. 10 → 0 Age: 50's B ring gel urticaria itches. Wheals started to be gradually remitted Sex: male formulation just after application and disappeared 1 application after 7 min. D26 1 μm rosacea Suffering from rosacea for 2 Tautness and tingling itches removed N/A Intractable Age: 30's B ring gel years. 2 months of facial just after application. Diffuse flare, after 2 yrs of Sex: formulation application of a steroid follicular papule and pustule remitted conventional female 1 application ointment exacerbated the after 1 min. After 1 week of 2 therapy symptoms, developing application/day, anti-inflammatory effect without telangiectasia, diffuse flare, was observed. Diffuse flare, follicular satisfactory follicular papule and pustule papule and pustule remarkably therapeutic on cheeks. Subjective improved. Itches and hot flush removed. effect. symptoms include tingling itches and tautness. D35 4 μm perioral 2 years of facial application of Diffuse flare and follicular papule N/A Age: 60's B ring gel dermititis a steroid ointment resulted in remarkably remitted 2 min after Sex: formulation circumoral telangiectasia, application. After 2 application/day for 2 female 1 application diffuse flare and follicular days flare improved and scales papule. Subjective symptoms removed. include heat sensation.

TABLE 8 Pruritus before and after Pre-application symptoms Post-application symptoms application Case Treatment Disease (severity) (severity) (VAS) Notes D36 2 μm rosacea Presented with circumoral Diffuse flare and follicular papule Age: 50's B ring gel telangiectasia, diffuse flare remarkably remitted 2 min after Sex: male formulation and follicular papule. application 1 application D37 1 μm acne Oily skin accompanied by Seborrhea remitted 2 min after N/A Faster and Age: 20's B ring gel vulgaris suppurative inflammation with application. Anti-inflammatory effect stronger Sex: female formulation many pustules and comedos remitted suppurative inflammation and anti- 1 application/ on lower jaw. pustules on lower jaw. Pustules reduced inflammatory day and dried 3 days after application, and effect than 3 days domedos disappeared. applying/ administrating conventional antibiotics. D38 Right cheek: acne Multiple follicular papules and Anti-inflammatory effect observed 3 min N/A Age: 40's 1 μm vulgaris pustules on both cheek. Oily after application, pustules and papules Sex: female B ring gel skin. started to be reduced and flattened, and formulation flare started to be removed. After 1 1 application/ week pustules and papules substantially day remitted and flare further remitted. After 2 weeks 2 weeks pustules, papules and flare almost disappeared. Left cheek: No change in symptoms 3 min after BNP gel application. After switching to B ring single formulation, similar effects as right application, cheek were obtained. then 1 μm B ring gel formulation 1 application/ day 2 weeks D39 Right cheek: acne Oily skin accompanied by Anti-inflammatory effect by B ring Age: 10's 2 μm vulgaris sppurative inflammation with application was observed on right cheek Sex: male B ring gel multiple red papules and in 3 min. Suppurative inflammation formulation pustules on both cheeks. remitted. 1 application Left cheek: No anti-inflammatory effect on gel formulation gel-applied left side. 1 application D40 4 μm acne Seborrheic skin with multiple Comedos faded in 5 min after Age: 10's B ring gel vulgaris comedos and follicular application. Oily skin improved to better Sex: female formulation papules/pustules on forehead. texture. Red papules and pustules 1 application reduced.

As shown in Tables 1 to 8, in all subjects, or in all dermatitis of any symptoms, emission or elimination of various symptoms of dermatitis was observed at the site where B ring gel formulation was applied.

Specifically, when B ring gel formulation was applied, remission or elimination of pruritus was observed immediately after the application. Generally in dermatitis, patients tend to scratch where pruritus was felt, potentially causing severer dermatitis. Such a remarkable remission or elimination of pruritus in this manner can consequently prevent aggravation of dermatitis.

In all cases, the effects of B ring gel formulation were superior to those with BNP gel formulation. Specifically, B ring gel formulation had faster-acting and longer-lasting effects as well as more potent antipruritic effect as compared with BNP gel formulation. It was confirmed that these effect cannot be obtained by the gel base.

3.2 Comparing B Ring Gel Formulation to Gel Base by Two-Sided Application

For subjects suffering various dermatitis, either B ring gel formulation or the gel base was applied to the affected site, and changes in symptoms before and after the application were observed (Table below).

TABLE 9 Case Applied site Treatment Disease Pre-application symptoms Post-application symptoms E1 Right B ring atopic Face presented with infiltrating Flare and pigmentation removed 2.5 min after Age: 20's dermatitis erythema, scales and application. Skin elasticity and desiccation Sex: female pigmentation, with intractable improved. Itches eliminated. Left gel base itches No remission of pigmentation, infiltrating erythema, scales and desiccation, with itches left. E2 Right B ring chronic Stiffness and hard lichenification Flare remitted soon after application. After Age: 30's eczema on both forearms, with infiltrating 3 min skin softened and moisturized. Itches Sex: male erythema and desiccation. eliminated. Left gel base No improvement in lichenification, itching and flare after 3 min. E3 Right B ring atopic Presented with hot flush and Flare started to fade soon after application, Age: 10's dermatitis puffiness. and was remarkably remitted after 3 min. Sex: female Left gel base No improvement in flushing after 3 min. E4 Right B ring chronic Stiffness and hard lichenification Flare remitted soon after application, Age: 40's eczema on both forearms, with infiltrating remarkably remitted after 3 min with improved Sex: male erythema, scales and hot infiltration and hot sensation, and softened sensation. skin. Left gel base Infiltration felt 3 min after application. Skin was hard with unimproved flare and hot sensation.

4. Confirming Cosmetic Effect

Effects of a cosmetic comprising B ring were evaluated from the results obtained using a gel formulations, a bath agent and a hair cosmetic product (shampoo or rinse) or body soap comprising the B ring to confirm their effects.

4.1.1 Confirming Skin-Improving Effect (Comparing B Ring Gel Formulation to BNP Gel Formulation)

The B ring gel formulation or BNP gel formulation was applied to subjects. The B ring gel formulation was applied on the right cheek or eye of each subject, the BNP gel formulation on the left cheek or eye, and changes in skin conditions were observed and compared (Table below). The changes in skin conditions were determined based on physical sensory evaluation by the subject and our objective observation as physicians.

TABLE 10 Case Applied site: method Pre-application symptoms Post-application symptoms F1 Right: B ring Presented with wrinkles, cheek 2 min after application, wrinkles on eye corner and forehead were Age: 69 gel formulation flabbiness, pigmentation on eye shallowed, skin was full. After 3 min, cheek flabbiness and skin Sex: female 0.5 μmol, corner. texture were improved, and pigmentation on eye corner was less 1 application prominent. After application for 3 days, large wrinkle on cheek was shallowed, flabbiness was improved, skin was firm, resilient, full and moisturized. Left: applied No improvement in wrinkles after 3 min. gel base F2 Right: B ring Presented with wrinkles, cheek 2 min after application, wrinkles at eye corner due to desiccation Age: 45 gel formulation flabbiness and desiccation. was less prominent, skin texture was improved, moisturized and Sex: female 0.5 μmol, softened. 1 application Left: applied No improvement in wrinkles and desiccation after 2 min. gel base F3 Right: B ring Presented with desiccation, light 30 sec after application, skin desiccation was improved, skin was Age: 21 gel formulation erythema, tautness and itch. moisturized and flexible with no tautness. After 1 min, flare was Sex: female 2 μmol, remitted and itches were removed. After 4 min, further softness was 1 application given and desiccation was remarkably improved. Left: applied No improvement observed in desiccation, itching and flare after gel base 4 min. F4 Right; B ring Presented with desiccation and 3 min after application, skin desiccation was improved, skin was Age: 24 gel formulation light erythema. moisturized and firm, with improved texture. Skin was flexible, Sex: female 2 μmol, soft and moisturized. 1 application Left: applied No improvement observed in desiccation and flare after 3 min. gel base F5 Right: B ring Presented with wrinkles on both 2 min after application, wrinkles on both corners of eyes were less Age: 28 gel formulation corners of eye, and desiccation. prominent, cheek dryness was improved, skin was moisturized and Sex: female 2 μmol, flexible. Perioral desiccation was remitted and skin became full 1 application and flexible. Left: applied No improvement observed in desiccation and wrinkles after 2 min gel base at gel-applied site on the left. F6 Right: B ring Presented with skin desiccation, 1 min after application, itches were remarkably remitted. After Age: 30 gel formulation oily skin and red papules around 3 min, skin desiccation and flare were improved, and skin was Sex: female 2 μmol, nose, and light erythema and moisturized and flexible with improved texture. Red papules 1 application rough skin texture. around nose almost disappeared, combination state of desiccation and oily skin was improved, and skin is kept healthy. Left: applied 3 min after application, desiccation, flare and itches are left, gel base and seborrhea and papules around nose were not remitted.

Table below summarizes examples in which B ring gel formulation was applied without applying two-sided. The changes in skin conditions were determined by similar method as described above.

TABLE 11 Case Method of application Pre-application symptoms Post-application symptoms G1 B ring gel formulation Presented with cheek flare, open After B ring application, flare was repressed, skin Age: 50 1 μmol pores, rough and hard skin texture. texture was improved, skin was moisturized and softened. Sex: female 1 application G2 B ring gel formulation Presented with wrinkles and 3 min after application at B ring-applied site, wrinkles Age: 49 2 μmol desiccation on forehead and eye on forehead and eye corner were shallowed and less prominent, Sex: female 1 application corner. skin desiccation was improved, and skin was moisturized and flexible. G3 B ring get formulation Presented with male skin 1 day after application at ring-applied site, skin desiccation Age: 40's 1 μmol desiccation and roughness. and roughness were improved, and razor burn was prevented. Sex: male 1 application G4 B ring gel formulation Presented with miliaria and 3 min after application at B ring-applied site, miliaria was Age: 50's 2 μmol pruritus accompanied with flare. prevented, flare and itches were remitted. Sex: male 1 application

Accordingly, it was shown that B ring of the present invention has a skin moisturizing effect and is capable of improving skin texture, and that it is also effective in improving skin, for instance in lightening (spots, dullness) and anti-aging (flabbiness, resilience, large wrinkles) as well as in improving mucosal condition such as roughened lips.

In all subjects, a remarkable improving effect in skin and its persistence was observed at the site where B ring gel formulation had been applied as compared to where BNP gel formulation had been applied. Specifically, effects as follows were observed at the site where B ring gel formulation had been applied as compared to where BNP gel formulation had been applied with significance: effects of improving skin, moisturizing skin, improving skin texture, providing skin with resilience and firmness, softening skin, suppressing skin desiccation, fading fine wrinkles, supplying and keeping skin with water and oil, shallowing and diminishing crow's feet, eliminating skin rawness, eliminating and suppressing itches, relieving skin roughness and maintaining skin condition healthy.

Particularly, in subjects in their 20's or 30's, remarkable effects such as improving or eliminating wrinkles, improving cheek flabbiness, and supplying and keeping skin with moisture, water and oil, and the effects such as giving skin firmness and tightness, lifting up cheeks, eliminating skin rawness and relieving skin roughness were observed at the site where B ring gel formulation had been applied as compared to the site where BNP gel formulation had been applied, and these effects were greater as compared to those at BNP gel-applied site.

Moreover, in subjects in their 40's and 50's, remarkable effects such as improving flabbiness, large wrinkles and desiccation, giving skin firmness, fading dullness and spots were exhibited immediately after the application of B ring gel formulation, and these effects were greater as compared to those upon applying BNP gel formulation.

The effects exhibited significantly faster in B ring gel formulation as compared to BNP gel formulation. Specifically, effects were confirmed in most cases such that wrinkles were vanished or started to vanish and faded and skin was made full and resilient. These effects lasted for at least several hours thereafter. No irritating symptoms were observed with application of B ring gel formulation.

FIG. 1 shows a photograph of the right-side face of a subject in her 60's after the application of B ring gel formulation. In this subject, effects were observed at 20 minutes after the application of B ring gel formulation such that the skin were moist, provided with moisture, complexion and softness, given firmness, improved texture, provided with resilience, and large wrinkles faded. This result was compared to BNP gel formulation as a comparative example, indicating that the cyclic peptide of the invention is not only effective on fine wrinkles but also is effective on wrinkles and flabbiness, and that it is also effective in fading spots and dullness.

4.1.2 Confirming Skin-Improving Effect (Comparing B Ring Gel Formulation to Gel Base)

In order to examine the presence or absence of the contribution by the gel base to the effects of B ring gel formulation and BNP gel formulation, three subjects were administered B ring gel formulation on their right face and the gel base on the left, and changes in skin conditions on the subject's faces were observed (Table below).

TABLE 12 Case Applied site: method Pre-application symptoms Post-application symptoms H1 Right: B ring gel Rough, stiff and dry skin texture. After 2 min skin was left soft. Age: 48 formulation Sex: female Left: applied gel base Skin was tingling. Open pores and rough skin texture were not improved. H2 Right: B ring Sensitive dry skin with roughness 1 min after application, skin tingling sensation Age: 26 and tingling stopped. After 2 min, B ring-applied side was felt Sex: female more moisturized. Left: applied gel base 1 min after application, skin was still tingling. After 2 min, the subject left that moisturizing effect is weak. H3 Right: Sensitive skin with tingling. Age: 30 Left: applied gel base Skin was tingling and temporarily red. Skin desiccation Sex: female was not improved. White scales were observed.

As a result, all subjects felt tingling sensation at the site where the gel base was applied, and dry feeling of skin was not improved. Also, in all subjects, no effect (e.g., improved skin texture or closed pores) was observed at the site where the gel base was applied. On the other hand, in all subjects, the skin area on which B ring gel formulation was applied has been moisturized and softened.

4.2 Observation on Mucosal Condition Upon Application of B Ring Gel Formulation.

The B ring gel formulation was applied to normal subjects who had symptoms of roughened lips, and changes in mucosal condition were observed (Table below). The changes in mucosal conditions were determined based on physical sensory evaluation by the subject and our objective observation as physicians.

TABLE 13 Case Applied site: method Pre-application symptoms Pos-application symptoms I1 B ring 1 μmol Cracks on mouth corner with pain. Skin became moisturized soon after application. After 2 min, Age: 20's 1 application Desiccation and wrinkles of lips. cracks on mouth corner were remitted. After 5 min, lips were Sex: female soft and no longer felt dry or tingling. I2 B ring 0.5 μmol Annoying lip desiccation. 3 min after application, lips became moisturized. Age: 30's 1 application Sex: female I4 B ring 2 μmol Annoying lip desiccation. After 2 min, lips were full and soft, and wrinkles were less Age: 30's 1 application prominent. Moisturized. Sex: female I3 B ring 2 μmol Lips were dry and tingling, After 2 min, lips were full, soft and moisturized, and wrinkles Age: 30's 1 application with wrinkles and scales were less prominent. Tingling sensation stopped. Sex: female I4 Right: B ring Cracks on mouth corner with pain. 2 min after application, cracks on mouth corner were remitted Age: 20's 1 μmol Dry and roughened lips with no more pain, and moisturized. Sex: male 1 application Left: gel base 2 min after application, cracks on mouth corner and pain were 1 application not improved

Accordingly, it was confirmed that the cyclic peptide of the invention has an activity of improving mucosal condition and it also has an immediate effect in such activity.

4.3 Observation on Scalp/Hair Condition Upon Application of B Ring Gel Formulation.

The B ring gel formulation was applied to normal subjects who had symptoms of scalp/hair, and changes in conditions of scalp and hair were observed (Table below). The changes in scalp/hair were determined based on physical sensory evaluation by the subject and our objective observation as physicians.

TABLE 14 Applied site: Case method Pre-application symptoms Post-application symptoms J1 B ring 0.5 μmol Presented with desiccation and After 2 min. itches were removed, scalp was relieved from Age: 40's 1 application itches of head. roughness and desiccation and moisturized. After that scalp Sex: male was prevented from desiccation and kept moisturized. J2 B ring 1 μmol Presented with erythema and itch Hair growth was stimulated, and an increase in hair volume Age: 50's 1 application/day, of head, and thinning and falling of was observed, and thinning of hair was improved. Hair was Sex: female 3 weeks hair. provided with moisture, flexibility and radiance. J3 B ring 1 μmol Presented with thinning and falling A decrease in falling hair was observed, showing an effect Age: 40's 1 application/day, of hair. of preventing alopecia. Hair growth was stimulated, and an Sex: female 5 days increase in hair volume was observed, and thinning of hair started to be improved. Hair was provided with moisture, flexibility and radiance. J4 B ring 2 μmol Presented with seborrheic dandruff After 2 min, itches were removed, scalp seborrhea and Age: 40's 1 application and itches. dandruff started to be improved. After that scalp became Sex: female and was kept clean. J5 B ring 2 μmol Presented with desiccation, mild After 1 min. itches were removed. After 2 min, scalp Age: 20's 1 application erythema and itches. seborrhea and dandruff started to be improved. After 4 min, Sex: male erythema and skin roughness of scalp was improved. After that skin roughness was prevented. J6 B ring 2 μmol Presented with scalp seborrhea, After 2 min, itches were removed, scalp seborrhea was improved. Age: 60's 1 application dandruff and itches. After that dandruff was removed, scalp became healthy and clean. Sex: male J7 B ring 1 μmol From the beginning of 5th month of Upon being applied to alopecia 5 month post partum, an effect Age: 30's To decalvant site 3rd pregnancy, significant hair loss was exhibited on hair loss in eyebrows and head where hair Sex: female on head and brows in eyebrows, lashes and scalp growth was stimulated, improving thinning of hair. of subject: 1 occurred, loosing almost all hair. application/day post partum, continued

Accordingly, BNP cyclic peptide of the present invention can provide and keep scalp and hair with moisture, give them moderate water and oil, improve and prevent desiccation, and clean scalp and hair. It is further effective for suppressing itch and dandruff of scalp and also effective for preventing thinning or loosing hair, for growing hair and promoting or stimulating hair growth, and forcing hair growth, and furthermore effective for an improvement in alopecia after illness or postnatal alopecia.

4.4 Observation on Skin Condition Upon Using Bath Agent Comprising B Ring

Subjects were given bath in 37 to 41° C. hot water comprising B ring dissolved at 0.01 μM (100 ml of 20 μM B ring solution dissolved in 200 L hot water) once a day for 14 days, and skin condition of the subjects was compared to the case of hot water alone (Table 14). The changes in skin conditions were determined based on physical sensory evaluation by the subject and our objective observation as physicians.

TABLE 15 Pre-application symptoms (

, cracks, chaps Case Treatment and eczema) Post-application symptoms Changes in subjective symptoms K1 B ring Eczema Gradual remission of itches after bath was remitted. Age: 30's addition erythema and skin Skin was moisturized. Sex: female desiccation. K2 Hot water Eczema Flare and desiccation Enhanced itches and desiccation Age: 30's exacerbated. after bath. Sex: female K3 BNP Eczema No remission of skin No subjective remssion of skin Age: 30's addition desiccation or erythema. desiccation at this concentration. Sex: female K4 B ring Chaps, rough skin Remission of desiccation. Remission of itch and pain. Skin Age: 60's addition of hands, itches became smooth Sex: female with tingling K5 Hot water Chaps and rough Exacerbation of desiccation. Exacerbation of itches and Age: 60's skin of hands tingling after some time after Sex: female bath. K6 B ring

 and itches Remission of

Remission of itches and improvement Age: 20's addition in skin texture. Sex: male K7 Hot water

 and itches No improvement in

No remission of itches. Age: 20's Sex: male K8 B ring Cracks and pain Cracks gradually became Relief of pain. Skin was moisturized. Age: 40's addition on sore shallower and improved Sex: female upon application K9 Hot water Cracks and pain No improvement in cracks. No remission of pain. Age: 40's on sore Sex: female

indicates data missing or illegible when filed

Accordingly, it was confirmed that the bath agent comprising B ring of the present invention has an ameliorating effect on miliaria, cracked or chapped skin, and also an improving effect on eczema. Furthermore, it was shown to improve desiccation, pain and itch of the skin, indicating that it is effective for improvement of skin condition.

4.5 Observation on Conditions of Scalp and Hair or Skin Upon Using Hair Cosmetics (Shampoo, Rinse) or Body Soap Comprising B Ring 4.5.1 A Shampoo Comprising B Ring was Mixed and Prepared in Composition as Follows.

TABLE 16 Name of ingredient (tradename) Content (%) A Sodium cocoylmethyltaurine solution 10.0 (NIKKOL CMT-30) Sodium laureth lactate (NIKKOL SBL-2N-27) 20.0 Lauryl betaine solution (NIKKOL AM-301) 10.0 Cocamide DEA 4.0 Antiseptic q.l. B Citric acid 0.1 Propylene glycol 2.0 Guar hydroxypropyl trimonium chloride 0.5 Water to fill up 100.0 C Water, B ring (B ring concentration 1 μM) 1 mL

A and B were dissolved with heat at 70° C. To A, B was added, stirred and mixed. C was added at 40 to 35° C. while being further stirred, and allowed to cool to room temperature as kept being stirred.

4.5.2 A Conditioner Comprising B Ring was Mixed and Prepared in Composition as Follows.

TABLE 17 Name of ingredient (tradename) Content (%) A Pentylene glycol 1.50 BG 3.50 Glycerin 1.00 Methyl paraben 0.20 Carbomer 0.20 EDTA-2Na 0.10 Water to fill up 100.0 B Composite emulsifier (NIKKOL Nikkomulese LC) 4.00 Methyl heptyl laurate 3.50 Squalane 0.50 Cetearyl alcohol 1.50 Macadamia nut oil 0.50 Avocado oil 0.50 Shea oil 0.50 Propyl paraben 0.10 C Water, B ring (B ring concentration 1 μM) 1 mL

A and B were dissolved with heat at 80° C. Then A was stirred by a homomixer while B was gradually added thereto, and the mixture was emulsified. C was further added to the mixture, and the mixture was allowed to cool to 35° C. as kept being stirred.

4.5.3 A Body Soap Comprising B Ring was Mixed and Prepared in Composition as Follows.

TABLE 18 Name of ingredient (tradename) Content (%) A Cocoyl glutamic acid TEA solution 30.0 Sodium trideceth-4 carboxylate 5.0 (KIKKOL ECTD-3 NEX) Sodium cocoamphoacetate solution 10.0 (KIKKOL AM-101) PEG-50 hydrogenated castor oil (KIKKOL HCO-50) 0.5 1,3-butylene glycol 5.0 Antiseptic q.l. B EDTA-2Na q.l. Water to fill up 100.0 C Water, B ring (B ring concentration 1 μM) 1 mL

A and B were dissolved with heat at 80° C. Then B was added to A while being stirred. C was further added to the mixture at 40 to 35° C. while kept being stirred, and the mixture was allowed to cool to room temperature as kept being stirred.

Subjects used shampoo, rinse or body soap prepared as above once a day for 14 days, and resulted conditions of scalp and hair or skin of the subjects were assessed (Table below). Changes in skin condition was assessed by dandruff of scalp. Changes in erythema was assessed by visual observation by the subject. Itches, moisturized feeling, combing, glow, resilience and elasticity of hair were assessed by subject's physical sensory evaluation.

TABLE 19 Case Treatment Pre-application symptoms Post-application symptoms Subjective changes L1 shampoo/ Presented with erythema and Remission of scalp Improvement in itches. Hair was Age: 60's treatment itches on scalp, and thinning erythema and a decrease moisturized and provided with Sex: male and falling hair. in hair falling. resilience, radiance and elasticity. L2 shampoo Presented with desiccation Improvement in dandruff Improvement in itches. Hair was Age: 20's and dandruff on scalp and desiccation of scalp. moisturized and provided with Sex: female accompanied with itches resilience, radiance and elasticity. Improved. With better combing. L3 body soap Dry and sensitive skin. Improvement in dry skin, improvement in itches after bath. Age: 60's relief of itching after bath. Sex: female L4 body soap Dry and sensitive skin. Improvement in dry skin, Improvement in itches after bath. Age: 60's relief of itching after bath. Sex: female L5 body soap Dry skin. Improvement in dry skin. Skin was moisturized after bath. Age: 20's Sex: male

Accordingly, the use of the hair agent comprising B ring of the present invention had an fast-acting improving effect on itch, erythema and desiccation of scalp. Moreover, it was shown to be effective in improving symptoms of thinning and falling hair when it was used continuously for at least 2 weeks. Furthermore, it was shown that the use of the body soap comprising B ring of the present invention has an improving effect on dry or sensitive skin.

5. Confirming Therapeutic Effect on Alopecia

Either B ring gel formulation or BNP gel formulation was applied, and their effects on symptoms associated to various alopecia were observed (Table below). The changes in symptoms were determined based on physical sensory evaluation by the subject and our objective observation as physicians.

TABLE 20 Case Treatment Disease Post-application symptoms A21 Right: B ring 1 μmol Male alopecia 7 weeks after application, hair became elastic and thick, Sex: male 1 application/ a drastic decrease in hair-falling Consequently, hair became Age: 50's day, continued. dense and thinning less prominent B ring-containing gel Left: BNP gel formulation exhibited more remarkable hair growing affect formulation 1 application/ day, continued. A5 B ring gel Male alopecia and 2 weeks after starting applying B gel formulation, the Sex: male formulation alopecia

existing soft hair in parietal area became thicker and Age: 50's 1 application/ accompanied with longer and turned black.. Hair got resilient and alestic. day onto head dandruff. Previous Newly grown terminal hairs remarkably decreased thinning decalvent site, use of

-containing area. Dandruff was suppressed overall, particularly in continued. drug caused strong parietal area.

 was eliminated. irritation, itches and erythema to the subject and its use was terminated A22 B ring gel Seborrhoic alopecia 2 weeks after application, thinning or hair was improved. Sex: female formulation accompanied with exhibiting no more showing-through of scalp confirming Age: 40's 2 application/ dandruff. hair-growing stimulation. Dandruff stopped, scalp seborrhea day onto was improved and pruritus was removed. Furthermore, scalp parietal and skin became clean. decalvant site.

indicates data missing or illegible when filed

TABLE 21 A10 B ring gel formulation Alopecia universalis. 2 weeks after starting application, on both sides of head, Sex: male 0.5 μmol Alopecia on whole head. regeneration of pores and growth of terminal hair were Age: 20's BNP gel formulation Previous therapy with confirmed, which were more remarkable and hair growth area 0.5 μmol carpronium chloride was wider on the B ring gel formulation-applied right side. and steroid pulse first Hair growth area kept increasing to the whole head. The exerted hair growth- density, elongation rate and thickness of hair were greater stimulating effect, but on the B ring gel formulation-applied right side. Upon caused complete hair increasing the concentration of application to 1 μmol, loss after 1 month. rate of growth and elongation were increased. A11 Right side of head decalvant Ophiasis and multiple Right forehead ophiasis site: 7 days after starting applying Sex: female site: B ring gel formulation alopecia. B ring gel formulation, a hair growth stimulation effect was Age: 40's 1 application/day for 1 week. observed and hair became thicker terminal hair, hair-growing 1 or 2 applications/week for 1 area kept increasing. year thereafter. Left BNP gel formulation-applied site: few soft hair observed, Left side of head decalvant whose growth was slow and in obviously smaller area. site: BNP gel formulation After continuing 1 or 2 applications/week for 1 year: 1 application/day for 1 week. Right forehead B ring gel formulation-applied site: hair- 1 or 2 applications/week for 1 growing area kept increasing and hair elongation rate was year thereafter. fast (FIG. 2). Left BNP gel formulation-applied site: hair growth was observed though its area was smaller as compared to the right side, and majority of hairs was thin and soft (FIG. 2).

TABLE 22 A12 Head decalvant site: Alopecia areata 1 week after application, remarkable stimulation and growth Sex: female B ring gel formulation of terminal hair was observed. Age: 20's 1 application/day, continued. A12 Subject's head decalvant Alopecia areata 1 week after application, remarkable stimulation, growth Sex: female site: B ring gel formulation and elongation of terminal hair was observed. Age: 30's 1 application/day, continued. A24 Right half of head decalvant Drug-induced Pre-application: 1 month after termination of anticancer Sex: female site: B ring gel formulation alopecia agent therapy. Bilateral severe diffuse alopecia on whole Age: 50's (1 μmol), 2 applications/ head. Thinning of hair on whole head. Hair was short and day for 2 weeks thin, mostly white. Left half of head decalvant 1 day after application at B ring-applied site, subjective site: BNP gel formulation hair elongation and growth stimulation were observed, and (1 μmol), 2 applications/ grown hairs were predominantly black (non-white). After 2 day for 2 weeks weeks, hair growth was observed at both sides. Hair were elongated with less falling hairs. Hair elongation rate is faster on B ring-applied site. Hairs are also thicker and denser, and contain more non-white hairs as compared to BNP-applied site. 3 weeks after application, remarkable growth of terminal hairs was observed.

5.2 Case Summary

In the cases of the subjects having female pattern alopecia, male pattern alopecia, alopecia universalis, ophiasis or alopecia areata multilocularis as described above, a significant decrease in falling hair was observed, and the area of terminal hair growth was expanded, and the growth of the terminal hair was also faster, when B ring gel formulation of the working example was applied compared with BNP gel formulation as a comparative control (See, FIG. 2). Addingly, the existing hairs obtained resilience and their elasticity was increased. In all cases, symptoms were significantly improved as compared to the cases when BNP gel formulation of the comparative example was applied. Accordingly, B ring gel formulation had superior effects on the symptoms described above as compared to BNP gel formulation.

In addition, the B ring gel formulation could improve seborrhea, clean scalp and suppress dandruff. It also prevents hairs from turning white, grows black hairs, and improves thinning of hair. Moreover, it exhibits an antipruritic effect within 10 minutes, demonstrating an excellent immediate effect. It could suppress ithes within 3 minutes after application in some cases.

6. Confirming Therapeutic Effect on Rhinitis 6.1 Cases

Either ring nasal drop, BNP gel nasal drop was sprayed, and their effects on rhinitis in the subject was observed (Table 17). The changes in symptoms were determined based on physical sensory evaluation by the subject and our objective observation as physicians.

TABLE 23 Case Treatment Disease Post-application symptoms (severity) R1 Right nasal cavity: Perennial chronic Right nasal cavity (B ring nasal drop-treated side): Subject felt breath goes through right Sex: female B ring nasal rhinitis and nasal cavity soon after application. After 30 sec, air goes through nasal cavity. After 2 Age: 20's formulation 0.1 ml, rhinorrhea. 20 or min. nose was clear and the subject could breathe comfortably. After 3 min, felt fresh 1 spray more blow/day and retained nasal discharge was removed. No discharge by blowing. A day after Left nasal cavity: application, no discharge from right nasal cavity. BNP nasal Left nasal cavity (BNP nasal drop-treated side): 3 min after application, the discharge formulation 0.1 ml, still retained in nasal cavity and drained by blowing. A day after application, mild nasal 1 spray obstruction and rhinorrhea symptoms in left nasal cavity with small amount of retained discharge. R2 Right nasal cavity: Rhinitis with severe Right nasal cavity (B ring nasal drop-treated side): Subject felt breath goes through right Sex: female B ring nasal nasal obstruction nasal cavity soon after application. After 30 sec, obstruction was improved, subject's Age: 30's formulation 0.1 ml, and feeling heavy. suffering in breathing was reduced in right nasal cavity. After 1.5 min, suffering in 1 spray Right nasal cavity breathing was removed in right nasal cavity and breath went though. A day after Left nasal cavity: has severer application, still no obstruction or rhinorrhea. Ths subject felt no irritation upon treatment BNP nasal obstruction symptom. in right nasal cavity formulation 0.1 ml, The subject feels dry Left nasal cavity (BNP nasal drop-treated side): 30 sec after application, obstruction of 1 spray and tingling by left nasal cavity was slightly improved, though the subject did not feel breath goes steroid nasal drops. through as in the right cavity. A day after application, obstruction or rhinorrhea in left nasal cavity were removed. The subject felt mild dryness in left nasal cavity. R3 Right nasal cavity: Perennial allergic Right nasal cavity: Subject felt breath goes through nasal cavity 1 min after application. Sex: female B ring nasal rhinitis with After 3 min nasal obstruction was improved, the subject could breathe through nose. Age: 20's formulation 0.1 ml, rhinorrhea and nasal After 5 min the subject could breathe comfortably through nose, with no nasal discharge 1 spray obstruction. by blowing. Right nasal cavity has severer obstruction with retention of nasal discharge.

TABLE 24 R4 Right nasal cavity: Chronic rhinitis with Right nasal cavity (B ring nasal drop-treated side): 1 min after treatment, nasal Sex: female B ring nasal severe rhinorrhea and obstruction was improved to some extent. After 2 min, obstruction was improved, the Age: 30's formulation 0.1 ml, nasal obstruction. subject could breathe through nose as usual, and rhinorrhea stopped. After 3 min, no 1 spray Application of steroid nasal discharge from the right nasal cavity by blowing. Left nasal cavity: nasal drops could not Left nasal cavity (BNP nasal drop-treated side): 1 min after treatment, nasal discharge BNP nasal improve symptoms when was retained in the left nasal cavity with no improvement in obstruction After 2 min, formulation 0.1 ml, they were severe. obstruction was improved to some extent, though discharge was retained, showing 1 spray rhinorrhea symptom. After 3 min, nasal discharge was not drained by blowing from left nasal cavity due to obstruction. After 8 min, nasal discharge was drained by blowing. R5 Right nasal cavity: Allergic rhinitis with Right nasal cavity (B ring nasal drop-treated side): 30 sec after treatment, obstruction Sex: female B ring nasal rhinorrhea as main was improved and the subject could breathe through nose. After 2 min, the subject Age: 20's formulation 0.1 ml, symptom and mild could easily breathe through nose. After 8 min, no nasal discharge was drained by 1 spray obstruction. blowing from the right nasal cavity. A day after treatment (after 26 hours), nasal Left nasal cavity: discharge was controlled, and breathing was easier through the right nasal cavity as BNP nasal compared to the left. formulation 0.1 ml, Left nasal cavity (BNP nasal drop-treated side): 2 min after treatment, obstruction was 1 spray still observed. After 8 min, small amount of discharge was drained by blowing. A day after treatment (after 26 hours), nasal discharge was controlled. R6 Right nasal cavity: Allergic rhinitis Right nasal cavity (B ring nasal drop-treated side): nasal obstruction was slightly Sex: female B ring nasal with both rhinorrhea improved soon after treatment. After 1 min, the subject could easily breathe though Age: 20's formulation 0.1 ml, and nasal obstruction nose. After 4 min, no nasal discharge was drained by blowing from the right nasal cavity. 1 spray having constant Nasal obstruction was resolved. After 10 min, pruritus was removed. A day after Left nasal cavity: obstruction and treatment (after 28 hours), no obstruction or rhinorrhea was observed. BNP nasal severe rhinorrhea. Left nasal cavity (BNP nasal drop-treated side): 1 min after treatment, nasal obstruction formulation 0.1 ml, 10-20 blow/day. was not improved. After 3 min, obstruction was still not improved and rhinorrhea was 1 spray Regular dose of oral observed. After 3 min, small amount of discharge was drained by blowing. After 10 min, anti-allergic drug obstruction was slightly improved but not resolved. After 2 hours, obstruction was did not sufficiently resolved and the subject could breathe though nose. A day after treatment (after 28 improve nasal hours), no obstruction or rhinorrhea was observed. obstruction and rhinorrhea. Steroid nasal spray causes mucosa pain.

TABLE 25 R21 Right nasal cavity: Rhinitis with continuous rhinorrhea Right nasal cavity (B ring nasal drop-treated side): Sex: female B ring nasal and sneeze. Nasal discharge was 30 sec after treatment, the subject could easily breathe Age: 20's formulation 0.1 ml, drained by facing down. through nose. After 1 min rhinorrhea stopped. No nasal 1 spray discharge by facing down. R8 Right nasal cavity: Chronic allergic rhinitis (with both Right nasal cavity (B ring nasal drop-treated side): Sex: female B ring nasal rhinorrhea and obstruction). The 1 min after treatment, obstruction was improved and the Age: 20's formulation 0.1 ml, subject regularly feels nasal subject could easily breathe through nose. The subject 1 spray + 1 spray obstruction. Application of steroid felt no irritation by treatment. The effect lasts for 1 20 min later nasal formulation causes the week thereafter, and rhinorrhea arid obstruction were Left nasal cavity: subject strong irritation. controlled. No nasal discharge by blowing. BNP nasal Accompanied with rhinorrhea and Commercial steroid nasal formulation caused a sharp formulation 0.1 ml, eye pruritus. irritation and recurrence of symptoms within 2 hours. 1 spray + 1 spray Left nasal cavity (BNP nasal drop-treated side): 20 min later R9 Right nasal cavity: Allergic rhinitis (with rhinorrhea). Right nasal cavity (B ring nasal drop-treated side): Sex: female B ring nasal The subject has rhinorrhea all day 2 min after treatment, the subject could breathe through Age: 30's formulation (1 μmol) and usually blows 20 or more times nose, with no irritation in the right nasal cavity. 0.1 ml, 1 spray a day. Application of steroid nasal After 4 min the subject could breathe through nose, with Left nasal cavity: formulation causes the subject no nasal discharge drained. BNP nasal strong dry feeling on mucosae of Left nasal cavity (BNP nasal drop-treated side): formulation (1 μmol) nasal cavity and pharynx. The 2 min after treatment, obstruction was improved and the 0.1 ml, 1 spray effect of the steroid nasal subject could breathe through nose. After 4 min, in the formulation lasts only 3-4 hours, left nasal cavity, the subject drained small amount of and after the effect ended, nasal discharge by blowing. In the right nasal cavity, sneezing and nasal discharge last the effect of the nasal treatment lasted for 4 hours. for several hours and need Blowing caused obstruction in the left nasal cavity but not frequent blowing. in the right. The subject felt that B ring nasal formulation is much better to the steroid nasal formulation regarding the effect on the symptoms, long-lasting and fast-acting effect, lower frequency of use required, and absence of irritating symptom upon use.

TABLE 26 R10 Right nasal cavity: Allergic rhinitis with rhinorrhea Right nasal cavity (B ring nasal drop-treated side): Sex: male B ring nasal and obstruction complicated with 2 min after nasal treatment, obstruction was improved Age: 30's formulation (1 μmol) rhinosinusitis. Obstruction in the and the subject could breathe through nose. The subject 0.1 ml, 1 spray right nasal cavity due to could breathe more comfortably in the right nasal cavity Left nasal cavity: rhinosinusitis is severer than the as compared to the left. The subject felt no irritation BNP nasal left and could not be improved by in the right nasal cavity. After 4 min, the subject could formulation (1 μmol) steroid nasal formulation, making very easily breathe, with no nasal discharge drained by 0.1 ml, 1 spray the subject incapable of breathing blowing. through nose. If left untreated, Left nasal cavity (BNP nasal drop-treated side): nasal discharge is drained from 4 min after nasal treatment, nasal obstruction was not nostrils spontaneously. resolved. Nasal discharge was decreased as compared to pre-treatment, though draining was still observed. R11 Right nasal cavity: Perennial rhinitis with rhinorrhea Right nasal cavity (B ring nasal drop-treated side): Sex: male B ring nasal and obstruction complicated with 2 min after nasal treatment, the subject could breathe Age: 20's formulation 0.1 ml, rhinosinusitis, having severe through nose. After 5 min, nasal discharge stopped, 1 spray obstruction. Very high frequency enabling the subject to breathe though nose. The subject Left nasal cavity: of blowing. These symptoms were felt no obstruction and was able to breathe through nose BNP nasal not improved by any previously throughout 1 week after nasal treatment. formulation 0.1 ml, used nasal formulation. Left nasal cavity (BNP nasal drop-treated side): 1 spray 5 min after nasal treatment, obstruction was not resolved, with nasal discharge present in the left nasal cavity. After 10 min. obstruction was not resolved, making the subject incapable of breathing through nose.

6.2 Case Summary

In all cases described above, both rhinorrhea and nasal obstruction were quickly improved or eliminated when B ring nasal drop of the working example had been applied as compared to the case when BNP nasal drop of comparative example had been applied. Accordingly, B ring nasal drop had faster effect than BNP nasal drop. Addingly, the effect of B ring nasal drop was more than equal to that of BNP nasal drop, and because the effect lasted for longer time period, it can suppressing recurrence of symptoms.

The following table summarizes information from the cases described above, within the range recognizable at the time when symptoms of nasal obstruction and rhinorrhea were improved.

TABLE 27 No resolution within within within within 3 min 5 min 10 min 10 min Resolution of rhinorrhea B ring nasal formulation 3 4 1 0 BNP nasal formulation 0 0 0 7 Resolution of nasal obstruction B ring nasal formulation 6 5 0 0 BNP nasal formulation 0 0 0 8

7. Analysis of Binding State of Human BNP and Type A Receptor

The superior pharmacological effects of the B ring-compound of the invention on various diseases relative to conventional BNP has been explained so far with reference to data based on respective clinical cases. Such pharmacological effects is also supported by the result of the conformation analysis of the compound performed by the inventors, which is explained by following experimental report for reference.

In order to investigate binding state of human BNP (BNP-32) and its receptor Type A receptor (NPR-A), an in silico analysis was performed by homology modeling using conformation. Swiss-Pdb viewer and SWISS-MODEL were used for modeling.

7.1 Template Structure

Firstly, prior to the above analysis, a template structure for investigation on binding state of human BNP and Type A receptor was selected. As this template structure, the conformation of the complex of rat NPR-A and rat ANP peptide (PDB ID: 1T34) were used. Rat NPR-A is a homodimer composed of A and B strands. The conformation of such rat NPR-A has been determined by X-ray crystalline structural analysis such that 21 residues from Cys7 to Arg 27 of rat ANP were bound. The conformation of rat NPR-A was obtained from the database of protein conformation, Protein Data Bank. Amino acid homology between human NPR-A and rat NPR-A is 85%.

7.2 BNP Peptide Model

In the present study, as BNP peptide model, a region from Cys10 to Arg30 in human BNP (BNP-32, SEQ ID NO: 13, Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Le u-Gly-Cys-Lys-Val-Leu-Arg-Arg-His), i.e., an amino acid sequence (SEQ ID NO: 14, Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg) was used. In the peptide model above, the amino acid sequence of B ring of the invention corresponds to a region from Cys10 to Cys26 of human BNP above, i.e., Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys (SEQ ID NO: 15).

7.3 Homology Modeling

In order to speculate the amino acid residues that are involved in binding of human NPR-A and human BNP, a complex model in which BNP peptide is bound to human NPR-A was constructed by homology modeling. Specifically, human BNP peptide model was constructed based on the template structure of rat ANP peptide, and human NPR-A model structure based on the template structure of rat NPR-A.

Next, residues that are involved in the interaction was speculated from the amino acid residues detected between human BNP peptide model and human NPR-A.

The results showed that BNP peptide model is bound to human NPR-A dimer being sandwiched between the A strand and B strand. Amino acids in the amino acid sequence, e.g., the amino acid sequence used as the model (SEQ ID NO: 14, Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg) are expressed as “amino acid (number)”. Namely, the number in parentheses denotes the relative position counted from the N-terminal of the amino acid sequence, for example, Phe2 indicates an amino acid that is the second Phe counted from the N-terminal of the peptide model.

In the constructed complex model, the presence of a hydrophobic bond by Phe2 side chain, a hydrogen bond by Phe2 main chain, and hydrogen bonds by side chains of Arg4, Met6, Arg8, Ser10 and Ser11 were speculated between BNP peptide model and the A strand of NPR-A. Phe2, Arg4, Met6, Arg8, Ser10 and Ser11 in BNP peptide model correspond to Phe11, Arg13, Met15, Arg17, Ser19 and Ser20 in human BNP respectively. Namely, this result suggests that these amino acid residues in human BNP is likely to be the residues that contribute to NPR-A activation.

7.4 Discussion

Thus, by in silico conformation analysis, it was speculated that, in human BNP, the amino acid residues that are present in its cyclic part are likely to contribute to NPR-A activation, whereas the amino acid residues present in the tail part are not. It was also suggested that BNP cyclic structure is a smaller molecule than BNP-32 and therefore capable of binding easily and quickly. In fact, a clinical application of a peptide having BNP cyclic structure confirmed that BNP cyclic structure provides faster therapeutic effect than BNP-32. This clinical result is consistent with that of the in silico analysis that BNP cyclic structure contributes to NPR-A activation and can bind to it more easily and quickly than BNP-32. Thus, BNP cyclic structure has a superior therapeutic effect than a general BNP peptide, and therefore is a different substance.

On the other hand, an NMR analysis revealed that ANP does not take any particular conformation in a solution where its conformation greatly wobbles; this is considered to be similar for BNP. Namely, in human BNP, assuming that the amino acid residues in the cyclic part contribute to its binding to human NPR-A, it is speculated that the amino acid residues in the tail part rather prevent human BNP from entering into the narrow BNP binding site sandwiched between A and B strands of human NPR-A due to its large wobbling. Therefore, it is considered that the cyclic part of human BNP is a relatively smaller molecule than conventionally known human BNP, enabling itself to enter into BNP binding site of NPR-A more easily and quickly. Moreover, it is speculated that the the cyclic part of human BNP has a higher affinity to human NPR-A than BNP-32. This supports the clinical outcomes described herein that the peptide having only the cyclic part of BNP exhibits therapeutic effect faster than BNP-32.

In order to speculate the effect of BNP cyclic structure (B ring) from non-human species on human Type A receptor, complex models of human NPR-A and BNP rings from pig, bird or rat were generated to surmise interaction. The results suggested that a sufficient effect of the invention can be expected by using BNP ring from non-human animal species (e.g., pigs, birds or rats), as long as it shows an affinity to human NPR-A.

8. Speculating Replaceable Amino Acid Residues in BNP Cyclic Moiety

Using the constructed model structure of the complex, replaceablity of amino acid residues other than those considered to be involved in the interaction was investigated.

Specifically, in order to investigate whether the peptide in which amino acid residue that is not assumed to be involved in the interaction has been replaced with another amino acid is capable of binding to NPR-A, a mutant model of BNP was generated to analyze the interaction. Swiss-Pdb viewer was used for modeling.

Specifically, amino acid residues Gly12, Lys14, Asp16, Ile18, Ser21, Ser22, Gly23, Leu24 and Gly25 in human BNP were targeted for the investigation on their replaceability with other amino acids in terms of following points:

-   -   No steric hindrance caused by binding to NPR-A. No interatomic         collision observed in the model structure of NPR-A and BNP.     -   No influence on electrostatic potential on surface.     -   No large increase in intramolecular energy value (no unnatural         angle or twist caused in intermolecular binding).     -   No non-naturally occurring hydrogen bond formed between the         strands of NPR-A and BNP and within BNP strand.     -   No cavity (cavity, niche) formation.

Among those described above, intramolecular energy was calculated by ComputeEnergy command of Swiss-Pdb viewer. Intramolecular energy was calculated from the sum of the length of binding, bond angle, twist and binding energy, etc. in the unit kilojoule/mol (Kj/mol).

The results of analysis indicated the presence of replaceable amino acid residues in Gly12, Lys14, Ile18, Ser21, Ser22, Leu24 and Gly25 among the amino acid residues of human BNP (Table below).

TABLE 28 human BNP Replaceable amino acid Gly12 Ala, Val, Ser, Thr Lys14 Arg Asp16 none Ile18 Val Ser21 Thr, Ala, Val, Gln, Leu, Ile, Met Ser22 Thr, Ala, Val Gly23 none Leu24 Ala, Val, Ile, Met Gly25 Ala, Ser

Accordingly, even when the replacement of the amino acid corresponding to those described in the table above took place in the cyclic part of human BNP, i.e., the peptide expressed by the Formula I-a, it was suggested that the peptide replaced in such a way exerts a similar effect as the peptide composed of the amino acid sequence expressed by Formula I-a.

9. Confirming Therapeutic Effect of the Cyclic Peptide with Amino Acid Replacement

Next, cyclic peptides with some replaced amino acid were subjected to following examination in order to confirm their effect. The methods for preparing the above cyclic peptide and confirming amino acid sequence of the prepared peptide were same as the method for preparing the cyclic peptide as described above and Mass spectroscopy method.

9.1 Confirming Effects on Scalp or Hair

The obtained cyclic peptide was prepared in purified water at 3, 15, 30, 100 or 500 μg/ml and applied to the affected site. Each of formulations A to E in the table indicates the concentration of cyclic peptide at 3, 15, 30, 100 or 500 μg/ml, respectively. The absence of the effect upon the application of purified water confirmed the effect was not a placebo effect.

TABLE 29 SEQ ID NO (amino acid For- sequence) of mu- the cyclic la- Diagnostic impression of hair peptide used ID Sex Age Cases tion Treatment and scalp after application SEQ ID NO 16:   S1 male 50's multiple alopecia C once a day After 1 day, fluffy hairs (CFVRKMDRISS areata started to grow. After 3 days, SSGLGC) obviously Hair started to grow, restoration and elongation were observed. Hair gained elasticity and resilience, thickened. Hair root regeneration confirmed.   S2 fe- 40's seborrheic derma- C once a day Iches were remitted immediately male titis male AGA after applicaation. After 3 minutes, seborrhea and erythema remitted. After 1 day, dandruff, redness and seborrhea of scalp improved, falling hairs reduced. After 7 days, hairs stimulated to grow, restored hair and hair growth confirmed.   S3 fe- 50's female AGA C once a day After 1 day, hair gained elasti- male city, resilience and increased volume.   S4 fe- 50's hair thinning C once a day After 5 days, hair stimulation male after anticancer and growth confirmed. Hair became agent therapy dense and thick.   S5 fe- 20's multiple alopecia C once a day Iches were improved immediately male areata after application. After 1 day, fluffy hairs started to grow. After 3 days, obviously Hair growth stimulated and restored elongation of hair observed, hair gained elasticity and resilience, thickened. Hair root regeneration observed.   S6 fe- 60's healthy skin  C once a day After 1 day, hair became fuller male (scalp) with increased volume. Hair gained elasticity and resilience. After 7 days, grown hairs were predominantly black over white hairs. White hairs turned black. SEQ ID NO 17:   S7 male 40's multiple alopecia B once a day After 3 days, remarkable terminal (CFGQKMDRISSS areata hair growth was observed. SGLGC)   S8 fe- 20's multiple alopecia B once a day After 5 days, newly born hair male areata roots and grown hairs were observed.   S9 male 30's male AGA B once a day After 2 or 3 days, hair gained elasticity and resilience. After 7 days, hair gained volume, restoration was confirmed.  S10 fe- 30's female alopecia C once a day Hair gained elasticity and resi- male lience, hair volume was increased on the night of first applica- tion. After 5 days, appearance of scalp through hair were improved. The effect lasted for 1 week after stopping application. SEQ ID NO 18:  S11 male 60's male alopecia B once a day After 1 week, hair gained thick- (CFGHKMDRISSS ness and elasticity, hair density SGLGC) was increased.  S12 male 70's healthy skin  C once a day After 1 day, hair became fuller (scalp) with increased volume. Hair gained elasticity and resilience. After 7 days, grown hairs were predominantly black over white hairs. White hairs turned black.  S13 fe- 40's alopecia areata B once a day After 5 days, hair started to male grow, elongation and growth of hair were observed.  S14 fe- 60's alopecia areata B once a day After 5 days, hair started to male grow, hair density was increased. SEQ ID NO 19:  S15 fe- 20's multiple alopecia B once a day After 5 days, hair started to (CFGRRMDRISSS male areata grow, elongation and restoration SGLGC) of hair were observed.  S16 fe- 50's hair thinning C once a day After 2 days, hair gained elasti- male after anticancer city and resilience volume of agent therapy hair increased  S17 male 60's male AGA C once a day After 1 day, hair gained elasti- city, resilience and increased volume. Hair started to grow, restoration and elongation were observed  S18 fe- 40's female alopecia D once a day After 3 days, scalp shown through male hair due to thinning got ob- secure. Reduced falling hairs.  S19 fe- 40's scalp seborrheic C once a day After 3 days, seborrhea and male dermatitis erythema were improved, so was dandruff. SEQ ID NO 20:  S20 male 50's male AGA C once a day After 1 day, hair gained elasti- (CFGRKLDRISSS city, resilience and increased SGLGC) volume.  S21 male  40's multiple alopecia C once a day After 3 days, hair density was areata increased. Hair started to grow and grew. SEQ ID NO 21:  S22 male 40's multiple alopecia C once a day After 3 days, hair started to (CFGRKIDRISSS areata grow at the site of alopecia SGLGC) areata and grew.  S23 male 40's male alopecia C 1 applica- After 3 days, hair started to tion/day on gain resilience, felt thick.  parietal After 7 days, parietal thinning male thin- were improved and hair volume ning area started to increase.  S24 fe- 40's female AGA C once a day After 1 day, hair gained elasti- male city, resilience and increased volume. Hair started to grow, restoration and elongation were observed.  S25 fe- 30's multiple alopecia C once a day After 1 day, hair started to  male areata grow. After 2 weeks, obvious lots of hairs started to grow, restor- ation, elongation and growth were observed.  S26 fe- 10's multiple ophiasis C once a day After 2 days, stimulated hair male growth was confirmed. After 7 days, obvious stimulated hair growth was observed, elongated and grown hair was also observed. SEQ ID NO 22:  S27 male 40's male AGA B 1 applica- After 2 or 3 days, hair gained (CFGRKMDRVSSS tion/day on elasticity and resilience. After SGLGC) forehead 7 days, hair gained volume, thinning restoration was confirmed. area  S28 male 20's multiple alopecia C once a day After 3 days, hair started grow areata and grew.  S29 fe- 60's female AGA C once a day After 1 day, hair gained elasti- male city, resilience and increased volume.  S30 fe- 70's female alopecia C once a day After 7 days, increased hair male volume. Appearance of scalp shown through hair got obsecure.  S31 fe- 20's ophiasis B once a day After 5 days, stimulated hair male confirmed along intractable hair- line, and elongation of hair observed.  S32 fe- 30's multiple alopecia C once a day After 1 day, hair started to  male areata grow. After 6 weeks, obvious stim- ulated hair growth, elongation and growth of hair were observed SEQ ID NO 23:  S33 male 30's alopecia areata C once a day After 3 days, stimulated and (CFGRKMDRIGSS universalis elongated hairs were observed SGLGC)  S34 fe- 60's hair thinning C once a day After 1 day, hair gained elasti- male after anticancer city and resilience. agent therapy  S35 fe- 50's female AGA C once a day After 1 day, hair gained elasti- male city, resilience and increased volume. Hair started to grow, restoration and elongation were observed.  S36 fe- 40's female alopecia B 1 applica- After 5 days, hair gained elasti- male tion/day on city, resilience and increased parietal volume. Appearance of scalp and fore- shown through hair got obsecure. head thin- ning area  S37 male   60's multiple alopecia C once a day After 1 day, stimulated hair was areata observed. After 3 days, applica- tion was stoped, but stimulation and elongation of hair contined. White hairs turned black again, black hairs started to grow.  S38 fe- 40's female alopecia B once a day After 7 days, hair gained volume, male seborrheic derma- appearance of scalp shown through titis hair got obsecure. Seborrhea of scalp was improved.  S39 fe- 10's ophiasis B once a day After 4 days, regardless of high- male ly intractable ophiasis, stimu- lated soft hairs were observed.   SEQ ID NO 24:  S40 fe- 10's alopecia areata A once a day After 7 days, stimulated and  (CFGRKMDRISAS male grown soft hairs were observed. SGLGC)  S41 fe- 50's female AGA C once a day After 1 day, hair gained elasti- male city, resilience and increased volume.  S42 male 30's multiple alopecia B once a day After 4 days, stimulation and areata elongation of hair observed.  S43 fe- 30's multiple alopecia C once a day After 1 day, hair started to  male areata grow. A number of stimulated, elongated and grown hairs were observed in 3 weeks. SEQ ID NO 25:  S44 male 60's multiple alopecia B once a day After 5 days, hairs started to (CFGRKMDRISSQ areata grow and grew. SGLGC)  S45 fe- 50's male AGA C once a day After 1 day, hair gained elasti- male city, resilience and increased volume. Hair started to grow, restoration and elongation were observed.  S46 fe- 60's multiple alopecia B once a day After 5 days, hair root regenera- male areata tion and hair growth stimulation were observed. Application con- tinued and a remarkable restora- tion, elongation and growth of hair were observed. Hair elonga- tion rate was fast.  S47 fe- 40's female alopecia C once a day After 1 day, hair gained elasti- male city and resilience. Application continued for 3 days and stopped, but hair kept elasticity and resilience. The effect lasted for 1 week. SEQ ID NO 26:  S48 fe- 30's female alopecia C once a day After 3 days, appearance of scalp (CFGRKMDRISSV male shown through hair got obsecure, SGLGC) hair gained elasticity, resili- ence and increased volume. Hairs were restored.  S49 male 30's male alopecia B once a day After 5 days, volume of hair increased. Hair gained elasticity and resilience.  S50 fe- 20's female AGA C once a day Volume of hair increased and male scalp does not show through after 3 days. The effect went on  S51 fe- 60's female AGA C once a day After 1 day, hair gained elasti- male city, resilience and increased volume. Hair started to grow, restoration and elongation were observed.  S52 male 20's multiple alopecia C once a day After 3 days, stimulated hair was areata observed After 7 days, further stimulation and growth of hair were con- firmed.  S53 fe- 30's multiple alopecia C once a day After 1 day, hair started to male areata grow. Terminal hairs started to grow, and restoration, elongation and growth of hair were observed in 2 weeks. SEQ ID NO 27:  S54 fe- 50's hair thinning B once a day After 5 days, hair got thick and (CFGRKMDRISSL male after anticancer dense. SGLGC) agent therapy  S55 male 50's male AGA C once a day After 1 day, hair gained elasti- city, resilience and increased volume. Hair started to grow, restoration and elongation were observed.  S56 male 20's multiple alopecia E once a day After 3 days, hair started to areata grow After 7 days, stimulation and growth of hair were confirmed. SEQ ID NO 28:  S57 fe- 40's female AGA C once a day After 1 day, hair gained elasti- (CFGRKMDRISSI male city, resilience and increased SGLGC) volume.  S58 male 20's multiple alopecia C once a day After 1 day, hair falling was re- areata duced and hair started to grow. After 7 days, remarkable stimula- tion, elongation and growth of hair were confirmed.  S59 male 20's multiple alopecia C once a day After 1 day, hair started to grow areata and falling hairs reduced. After 4 days, application was stoped, but stimulation, elongation and restoration of hair persisted.  S60 fe- 50's healthy skin  C once a day After 1 day, hair became fuller male (scalp) with increased volume. Hair gained elasticity and resilience. After 7 days, grown hairs were predominantly black over white hairs. White hairs turned black.  S61 fe- 40's multiple alopecia C once a day After 3 days, hairs started to male areata grow and grew.  S62 fe- 40's alopecia areata B once a day After 5 days, stimulated hair male growth was confirmed. After 2 weeks, further growth of terminal hair, elongation and growth of hair were confirmed all over alopecia site. SEQ ID NO 29:  S63 fe- 40's female alopecia C once a day After 4 days, hair gained elasti- (CFGRKMDRISSM male city, resilience and increased SGLGC) volume.  S64 male 40's healthy skin  C once a day After 1 day, hair volume was in- (scalp) creased. Hair gained elasticity and resilience.  S65 male 40's male AGA C once a day Hair gained resilience and volume after 3 days. Application con- tinued for 1 week, and the effect persisted for 1.5 months there- after.  S66 male 30's male alopecia C 2 applica- After 3 days, hair gained elasti- tion/day city, resilience and increased for 3 days volume at thinning area on fore- head. After a few days, hair growth stimulation and hair growth were confirmed.  S67 fe- 70's female alopecia C once a day After 2 days, hair gained elasti- male city and resilience. After 5 days, volume of hair increased. It was confirmed that the stimu- lated hairs are predominantly black over white hair. SEQ ID NO 30:  S68 fe- 20's alopecia areata B once a day After 7 days, stimulated hair (CFGRKMDRISSS male ophiasis growth was confirmed. Even 2 VGLGC) weeks after stopping application, hair growth stimulation and re- storation persisted, and there were little falling hairs.  S69 male 40's male AGA B once a day After 2-3 days, hair gained elasticity and resilience. After 7 days, hair gained volume, re- storation was confirmed.  S70 male 30's alopecia univer- B once a day After 3 days, hairs started to salis grow and grew.  S71 fe- 30's multiple alopecia C once a day After 1 day, stimulated hair male areata growth was confirmed. SEQ ID NO 31:  S72 fe- 20's alopecia areata C once a day After 7 days, stimulated hair (CFGRKMDRISSS male ophiasis growth was confirmed. Even 2 SRLGC) weeks after stopping application, hair growth stimulation and re- storation persisted, and falling hairs significantly decreased  S73 male 40's healthy skin C once a day After 1 day, hair volume was in- (scalp) creased. Hair gained elasticity and resilience.  S74 male 30's alopecia areata C 2 applica- After 3 days, stimulation and universalis tion/day elongation of hair were con- for 3 days firmed.  S75 fe- 60's multiple alopecia B once a day After 5 days, hairs stimulated to male grow, restored hair and hair growth were confirmed. It was confirmed that hair elongation rate is fast. SEQ ID NO 32:  S76 male 30's male alopecia B once a day After 7 days, hair gained elasti- (CFGRKMDRISSS city. Hair started to grow, and SGMGC) volume of hair on parietal fore- head was increased.  S77 fe- 60's female AGA C once a day After 1 day, hair gained elasti- male city, resilience and increased volume. Hair started to grow, restoration and elongation were observed.  S78 male 40's alopecia univer- C once a day After 3 days, hair stimulation  salis and growth were confirmed  S79 fe- 50's hair thinning C once a day After 2 days, hair gained elasti- male after anticancer city, resilience and increased agent therapy volume. Appearance of scalp shown through hair got obsecure. Hair increase was confirmed. SEQ ID NO 33:  S80 fe- 50's multiple alopecia B once a day After 7 days, stimulated hair (CFGRKMDRISSS male areata growth was confirmed. SGIGC)  S81 male 60's healthy skin C once a day After 1 day, hair became fuller (scalp with increased volume. Hair gained elasticity and resilience. After 7 days, grown hairs were predominantly black over white hairs. White hairs turned black.  S82 fe- 40's multiple alopecia B once a day After 5 days, hair root regenera- male areata tion and hair growth stimulation were confirmed.   S83 male 60's alopecia areata C once a day After 1 day, apparent hair growth stimulation and restoration were confirmed. After 4 days, hair elongation were confirmed. SEQ ID NO 34:  S84 fe- 50's hair thinning B once a day After 5 days, thin, fuzzy hairs (CFGRKMDRISSS male after anticancer along hairline got thicker and SGVGC) agent therapy denser.  S85 male 50's male AGA C once a day After 1 day, hair gained elasti- city, resilience and increased volume. Hair started to grow, restoration and elongation were observed.  S86 male 40's male AGA C once a day Hair gained resilience and volume after 3 days. Application con- tinued for 1 week, and the effect persisted for 1.5 months there- after.  S87 male 30's male alopecia C once a day After 3 days, hair gained resili- ence and volume of hair increased in parietal thinning area  S88 male 20's multiple alopecia C once a day After 1 day, hairs started to areata grow and grew. SEQ ID NO 35:  S89 fe- 40's hair thinning B once a day After 5 days, hair gained elasti- (CFGRKMDRISSS male after anticancer city, resilience, thickness, and SGAGC) agent therapy increased volume.  S90 male 20's multiple alopecia C once a day After 3 days, stimulated hair  areata growth was confirmed.  S91 fe- 50's female AGA C once a day After 1 day, hair gained elasti- male city, resilience and increased volume. Hair started to grow, restoration and elongation were observed.  S92 fe- 60's multiple alopecia B once a day After 5 days, hair root regenera- male areata tion and hair growth stimulation were observed. Then, remarkable restoration, elongation and  growth of hair were observed.  S93 fe- 30's multiple alopecia C once a day After 1 day, stimulated hair was male areata observed. After 2 weeks, there is remarkable elongation of hair marginal area around large de- calvant spot, and new hair start- ed to grow in center part. SEQ ID NO 36:  S94 male 30's alopecia univer- C once a day After 4 days, hair stimulation (CFGRKMDRISSS salis and growth were confirmed. SGLSC)  S95 male 40's healthy skin C once a day After 1 day, hair volume was in- (scalp) creased. Hair gained elasticity and resilience.  S96 male 50's seborrheic derma- C once a day After 3 days, seborrhea and  titis erythema were improved.  S97 male 50's male alopecia B once a day After 5 days, hair gained elasti- city, resilience and increased volume. SEQ ID NO 37:  S98 fe- 40's hair thinning B once a day After 5 days, hair gained elasti- (CFGRKMDRISSS male after anticancer city, resilience, thickness and SGLAC) agent therapy increased volume.  S99 male 40's male AGA B 1 applica- After 2 or 3 days, hair gained tion/day on elasticity and resilience. After forehead 7 days, hair gained volume, re- thinning  storation was confirmed. area S100 male 20's multiple alopecia C once a day After 5 days, stimulated hair areata growth was confirmed. S101 fe- 20's alopecia areata A once a day After 10 days, hair growth stimu- male lation elongation were confirmed. SEQ ID NO 38: S102 fe- 50's female AGA C once a day After 1 day, hair gained elasti- (CFARKMDRISSS male city, resilience and increased SGLGC) volume. Hair started to grow, restoration and elongation were observed. S103 fe- 50's dandruff, itches C once a day Iches stopped in 3 minutes,  male dandruff was reduced, redness in scalp was significantly improved. S104 fe- 10's eclopic multple C once a day After 3 days, stimulated hair male alopecia growth was confirmed in eyebrows. SEQ ID NO 39: S105 male 10's alopecia univer- C once a day After 3 days, stimulated hair (CFSRKMDRISSS salis growth was confirmed. SGLGC) S106 fe- 50's dandruff, itches C once a day Iches stopped in 1 minute, dan- male druff was reduced, redness in scalp was significantly improved. S107 fe- 60's healthy skin C once a day After 1 day, hair became fuller male (scalp) with increased volume. Hair gained elasticity and resilience. After 7 days, grown hairs were predominantly black over white hairs. White hairs turned black. S108 fe- 60's multiple alopecia C once a day After 3 days, stimulated hair male areata growth was confirmed. More black hairs started to grow than white hairs, and hair elongation rate was fast. SEQ ID NO 40: S109 male 60's male AGA C once a day After 1 day, hair gained elasti- (CFTRKMDRISSS city, resilience and increased SGLGC) volume. Hair started to grow, restoration and elongation were observed. S110 male 20's multiple alopecia C once a day After 4 days, stimulated hair areata growth was confirmed C once a day SEQ ID NO 41: S111 fe- 50's female AGA C once a day After 1 day, hair gained elasti- (CFGRKMDRISST male city, resilience and increased SGLGC) volume. Hair started to grow, retoration and elongation were observed. S112 male 20's multiple alopecia C once a day After 4 days, stimulated hair areata growth was confirmed. SEQ ID NO 42: S113 male 30's multiple alopecia C once a day After 3 days, stimulated hair (CFGRKMDRISSA areata growth was confirmed. SGLGC) S114 male 40's healthy skin C once a day After 1 day, hair volume was in- (scalp) creased. Hair gained elasticity and resilience. S115 fe- 60's multiple alopecia C once a day After 3 days, stimulated hair male areata growth was confirmed. Black hairs started to grow and hair elonga- tion was fast SEQ ID NO 43: S116 male 40's male AGA B 1 applica- After 2-3 days, hair gained (CFGRKMDRISSS tion/day on elasticity and resilience. After TGLGC) forehead 7 days, hair gained volume, re- thinning storation was confirmed area S117 male 20's multiple alopecia D once a day After 3 days, stimulated hair areata growth was confirmed. SEQ ID NO 44: S118 fe- 60's female AGA C once a day After 1 day, hair gained elasti- (CFGRKMDRISSS male city, resilience and increased AGLGC) volume. Hair started to grow, restoration and elongation were observed S119 fe- 20's multiple alopecia C once a day After 3 days, stimulated hair male areata growth was confirmed. SEQ ID NO 45: S120 male 40's male AGA C once a day After 1 day, hair gained elasti- (CFSRRMDRISSS city, resilience and increased SGLGC) volume. Hair started to grow, restoration and elongation were observed. S121 fe- 40's healthy skin C once a day After 1 day, hair volume was in- male (scalp) creased. Hair gained elasticity and resilience. SEQ ID NO 46: S122 fe- 60's female AGA C once a day After 1 day, hair gained elasti- (CFSRKMDRISST male city, resilience and increased SGLGC) volume. Hair started to grow, restoration and elongation were observed. S123 fe- 60's healthy skin C once a day After 1 day, hair became fuller male (scalp) with increased volume. Hair gained elasticity and resilience. After 7 days, grown hairs were predominantly black over white hairs. White hairs turned black. SEQ ID NO 47: S124 male 40's healthy skin C once a day After 1 day, hair volume was in- (CFSRKMDRISSS (scalp) creased. Hair gained elasticity TGLGC) and resilience. S125 fe- 50's female AGA C once a day After 1 day, hair gained elasti- male city, resilience and increased volume. Hair started to grow, re- storation and elongation were observed. SEQ ID NO 48: S126 fe- 40's multiple alopecia B once a day After 5 days, hair root regenera- (CFSRKMDRISSS male areata tion and hair growth stimulation SGIGC) were confirmed S127 fe- 60's healthy skin C once a day After 1 day, hair became fuller male (scalp) with increased volume. Hair gained elasticity and resilience. After 7 days, grown hairs were predominantly black over white hairs. White hairs turned black S128 fe- 50's female AGA C once a day After 1 day, hair gained elasti- male city, resilience and increased volume. Hair started to grow, restoration and elongation were observed. SEQ ID NO 49: S129 fe- 60's female AGA C once a day After 1 day, hair gained elast- (CFSRKMDRISSS male city, resilience and increased SGLAC) volume. S130 fe- 60's alopecia areata B once a day After 5 days, hair started to male grow and hair density was in- creased S131 fe- 40's healthy skin C once a day After 1 day, hair volume was in- male (scalp) creased. Hair gained elasticity and resilience. SEQ ID NO 50: S132 male 50's male AGA C once a day After 1 day, hair gained elasti- (CFGRRMDRISST city, resilience and increased SGLGC) volume. Hair started to grow, re- storation and elongation were observed. S133 male 40's healthy skin C once a day After 1 day, hair volume was in- (scalp) creased. Hair gained elasticity and resilience. SEQ ID NO 51: S134 male 40's male AGA B 1 applica- After 2 or 3 days, hair gained (CFGRRMDRISSS tion/day on elasticity and resilience. After TGLGC) forehead 7 days, hair gained volume, re- thinning storation was confirmed. area S135 male 50's healthy skin C once a day After 1 day, hair became fuller (scalp) with increased volume. Hair gained elasticity and resilience. SEQ ID NO 52: S136 male 60's male AGA C once a day After 1 day, hair gained elasti- (CFGRRMDRISSS city, resilience and increased SGIGC) volume. Hair started to grow, re- storation and elongation were observed. S137 fe- 40's multiple alopecia B once a day After 5 days, hair root regenera- male areata tion and hair growth stimulation were confirmed. S138 fe- 60's healthy skin C once a day After 1 day, hair became fuller male (scalp) with increased volume. Hair gained elasticity and resilience. After 7 days, grown hairs were predominantly black over white hairs. White hairs turned black. SEQ ID NO 53: S139 fe- 40's female AGA C once a day After 1 day, hair gained elasti- (CFGRRMDRISSS male city, resilience and increased SGLAC) volume. S140 male 40's healthy skin C once a day After 1 day, hair volume was in- (scalp) creased. Hair gained elasticity and resilience. SEQ ID NO 54: S141 male 40's male AGA B 1 applica- After 2 or 3 days, hair gained (CFGRKMDRISST tion/day on elasticity and resilience. After SGIGC) forehead 7 days, hair gained volume, re- thinning storation was confirmed. area S142 fe- 50's healthy skin C once a day After 1 day, hair became fuller male (scalp) with increased volume. Hair gained elasticity and resilience. After 7 days, grown hairs were predominantly black over white hairs. White hairs turned black. SEQ ID NO 55: S143 male 50's male AGA C once a day After 1 day, hair gained elasti- (CFGRKMDRISST city, resilience and increased SGLAC) volume. Hair started to grow, re- storation and elongation were observed. S144 fe- 60's healthy skin C once a day After 1 day, hair became fuller male (scalp) with increased volume. Hair  gained elasticity and resilience. After 7 days, grown hairs were predominantly black over white hairs. White hairs turned black. SEQ ID NO 56: S145 male 40's male AGA C once a day After 1 day, hair gained elasti- (CFGRKMDRISSS city, resilience and increased TGIGC) volume. Hair started to grow, re- storation and elongation were observed. S146 male 40's healthy skin C once a day After 1 day, hair volume was in- (scalp) creased. Hair gained elasticity and resilience. SEQ ID NO 57: S147 male 50's male AGA C once a day After 1 day, hair gained elasti- (CFGRKMDRISSS city, resilience and increased TGLAC) volume. Hair started to grow, re- storation and elongation were observed. S148 male 40's healthy skin C once a day After 1 day, hair volume was in- (scalp) creased. Hair gained elasticity and resilience SEQ ID NO 58: S149 fe- 40's female AGA C once a day After 1 day, hair gained elasti- (CFGRKMDRISSS male city, resilience and increased SGIAC) volume. S150 fe- 40's healthy skin C once a day After 1 day, hair volume was in- male (scalp) creased. Hair gained elasticity and resilience. SEQ ID NO 59: S151 male 50's male AGA C once a day After 1 day, hair gained elasti- (CFSRRMDRISST city resilience and increased SGLGC) volume. Hair started to grow, re- storation and elongation were observed. S152 male 40's healthy skin C once a day After 1 day, hair volume was in- (scalp) creased. Hair gained elasticity and resilience. SEQ ID NO 60: S153 male 60's male AGA C once a day After 1 day, hair gained elasti- (CFSRRMDRISSS city, resilience and increased TGLGC) volume. Hair started to grow, re- storation and elongation were observed. S154 male 40's healthy skin C once a day After 1 day, hair volume was in- (scalp) creased. Hair gained elasticity and resilience. SEQ ID NO 61: S155 fe- 60's female AGA C once a day After 1 day, hair gained elasti- (CFSRRMDRISSS male city, resilience and increased SGIGC) volume. Hair started to grow, re- storation and elongation were observed. S156 male 60's healthy skin C once a day After 1 day, hair became fuller (scalp) with increased volume. Hair gained elasticity and resilience. After 7 days, grown hairs were predominantly black over white hairs. White hairs turned black. SEQ ID NO 62: S157 male 50's male AGA C once a day After 1 day, hair gained elasti- (CFSRRMDRISSS city, resilience and increased SGLAC) volume. Hair started to grow, re- storation and elongation were observed. S158 male 40's healthy skin C once a day After 1 day, hair volume was in- (scalp) creased. Hair gained elasticity and resilience. SEQ ID NO 63: S159 male 60's male AGA B 1 applica- After 2 or 3 days, hair gained (CFSRKMDRISST tion/day  elasticity and resilience. After SGIGC) on forehead 7 days, hair gained volume, re- thinning storation was confirmed. area S160 male 40's healthy skin C once a day After 1 day, hair volume was in- (scalp) creased. Hair gained elasticity and resilience. SEQ ID NO 64: S161 male 60's healthy skin C once a day After 1 day, hair became fuller (CFSRKMDRISSS (scalp) with increased volume. Hair TGIGC) gained elasticity and resilience. After 7 days, grown hairs were predominantly black over white hairs. White hairs turned black. S162 fe- 50's female AGA C once a day After 1 day, hair gained elasti- male city, resilience and increased volume. Hair started to grow, re- storation and elongation were observed SEQ ID NO 65: S163 male 40's male AGA B 1 applica- After 2 or 3 days, hair gained (CFSRKMDRISSS tion/day on elasticity and resilience. After TGLAC) forehead 7 days, hair gained volume, re- thinning storation was confirmed. area S164 male 40's healthy skin C once a day After 1 day, hair volume was in- (scalp) creased. Hair gained elasticity and resilience. SEQ ID NO 66: S165  male 60's male AGA C once a day After 1 day, hair gained elasti- (CFSRKMDRISSS city, resilience and increased SGIAC) volume. Hair started to grow, re- storation and elongation were observed. S166 male 40's healthy skin C once a day After 1 day, hair volume was in- (scalp) creased. Hair gained elasticity and resilience. SEQ ID NO 67: S167 male 40's male AGA B 1 applica- After 2-3 days, hair gained  (CFGRRMDRISST tion/day on elasticity and resilience. After SGIGC) forehead 7 days, hair gained volume, re- thinning storation was confirmed. area S168 fe- 60's healthy skin C once a day After 1 day, hair became fuller male (scalp) with increased volume. Hair gained elasticity and resilience. After 7 days, grown hairs were predominantly black over white hairs. White hairs turned black. SEQ ID NO 68: S169 fe- 40's female AGA C once a day After 1 day, hair gained elasti- (CFGRRMDRISST male city, resilience and increased SGLAC) volume. S170 male 40's healthy skin C once a day After 1 day, hair volume was in- (scalp) creased. Hair gained elasticity and resilience. SEQ ID NO 69: S171 male 60's male AGA B 1 applica- After 2 or 3 days, hair gained (CFGRRMDRISSS tion/day on elasticity and resilience. After TGIGC) forehead 7 days, hair gained volume, re- thinning storation was confirmed. area S172 male 30's multiple alopecia C once a day After 1 day, hairs started to areata grow and grew. S173 male 60's healthy skin C once a day After 1 day, hair became fuller (scalp) with increased volume. Hair gained elasticity and resilience. After 7 days, grown hairs were predominantly black over white hairs. White hairs turned black. SEQ ID NO 70: S174 fe- 50's female AGA C once a day After 1 day, hair gained elasti- (CFGRRMDRISSS male city, resilience and increased TGLAC) volume. S175 male 40's healthy skin C once a day After 1 day, hair volume was in- (scalp) creased. Hair gained elasticity and resilience. SEQ ID NO 71: S176 male 60's male AGA C once a day After 1 day, hair gained elasti- (CFSRRMDRISST city, resilience and increased SGIGC) volume. Hair started to grow, re- storation and elongation were observed. S177 male 20's multiple alopecia C once a day After 1 day, hairs started to areata grow and grew. S178 male 60's healthy skin C once a day After 1 day, hair became fuller (scalp) with increased volume. Hair gained elasticity and resilience. After 7 days, grown hairs were predominantly black over white hairs. White hairs turned black. SEQ ID NO 72: S179 male 40's male AGA B 1 applica- After 2 or 3 days, hair gained (CFSRRMDRISST tion/day on elasticity and resilience. After SGLAC) forehead 7 days, hair gained volume, re- thinning storation was confirmed. area S180 male 40's healthy skin C once a day After 1 day, hair volume was in- (scalp) creased. Hair gained elasticity and resilience. SEQ ID NO 73: S181 fe- 40's female AGA C once a day After 1 day, hair gained elasti- (CFSRRMDRISSS male city, resilience and increased TGIGC) volume. S182 fe- 40's alopecia areata B once a day After 5 days, hair started to male grow and hair density was in- creased. S183 fe- 60's healthy skin C once a day After 1 day, hair became fuller male (scalp) with increased volume. Hair gained elasticity and resilience. After 7 days, grown hairs were predominantly black over white hairs. White hairs turned black. SEQ ID NO 74: S184 male 60's male AGA C once a day After 1 day, hair gained elasti- (CFSRRMDRISSS city, resilience and increased TGLAC) volume. Hair started to grow, re- storation and elongation were observed. S185 male 40's healthy skin C once a day After 1 day, hair volume was in- (scalp) creased. Hair gained elasticity and resilience. SEQ ID NO 75: S186 male 40's male AGA B 1 applica- After 2 or 3 days, hair gained (CFSRRMDRISSS tion/day on elasticity and resilience. After SGIAC) forehead 7 days, hair gained volume, re- thinning storation was confirmed. area S187 fe- 40's multiple alopecia B once a day After 5 days, hair root regenera- male areata tion and hair growth stimulation were confirmed. S188 fe- 50's healthy skin C once a day After 1 day, hair became fuller male (scalp) with increased volume. Hair gained elasticity and resilience. After 7 days, grown hairs were predominantly black over white hairs. White hairs turned black. In SEQ ID NOS 16-75. 1st Cys and 17th Cys from left form a disulfide bond.

The result described above demonstrated that the cyclic peptide of the invention exhibits a similar effect as the cyclic peptide expressed by Formula I-a even if some amino acids of the cyclic peptide have been replaced.

Specifically, it has effects of preventing hair loss at the applied site, stimulating hair growth or promoting hair growth, giving hair resilience and elasticity, increasing volume of hair and dramatically decreasing falling hairs when it was applied to the site of alopecia or hair growth stimulation, etc. It also has effects of nourishing hair, promoting hair growth stimulation, improving/preventing thinning of hair at the applied site. In this case, the stimulated hair tends to become a terminal (non-white) hair. Moreover, these effect exhibit significantly faster as compared to other active agents conventionally used in alopecia therapeutics such as BNP such that stimulated hair growth can be confirmed one day after only one application even in the case of an intractable alopecia areata multilocularis or ophiasis. In addition, resulting effects persists remarkably even after terminating application and the stimulated hair keep growing. In the case of AGA or healthy subject it exert immediate effect of providing hair with resilience and elasticity on the following day, increasing the volume, and dramatically decreasing falling hairs, which effects last even after terminating application. It also has an improving and preventing effect on dermatitis. Therefore, it can improve or prevent skin inflammation associated with alopecia. Such effect is advantageous in cases where alopecia has been exacerbated due to skin condition of the applied site (e.g., scalp). Moreover, the external preparation of the present invention exerts a moisturizing and skin texture-improving effects at the applied site when being applied to skin as described above. It can remove and suppress dandruff and itching, while giving moisture to hair and scalp, improving and preventing dryness and keeping hair and scalp healthy. It also can improve seborrhea. On the other hand, when it is used as an alopecia therapeutic/prophylactic, it can be used for the purpose of treating or preventing one or more of applicable alopecia including, for example, those described below, without being particularly limited thereto.

(Acquired Alopecia)

(i) Alopecia without accompanying scarring or skin lesion (alopecia areata, male pattern alopecia, seborrheic alopecia, alopecia pityroides, female pattern alopecia, gestational alopecia, malignant alopecia, senile alopecia, alopecia totalis, alopecia areata multilocularis, ophiasis, drug-induced alopecia, cancer chemotherapy-induced alopecia and radiation exposure-induced alopecia, traumatic/mechanical alopecia, malnutrition/metabolic disorder associated alopecia, endocrine dysfunction associated alopecia and telogen effluvium (post partum alopecia, alopecia after high fever)). (ii) Alopecia observed in skin lesion or pathologic skin (infection-induced alopecia, tumor-induced alopecia, inflammation-induced alopecia). (iii) Scarring alopecia (skin infection-induced alopecia, alopecia induced by infiltration of inflammatory cells).

(Congenital Alopecia)

Diffuse alopecia, congenital atrichia, alopecia in hereditary syndromes, localized alopecia, phakomatosis, aplasia cutis, congenital alopecia triangularis.

It exerts an excellent effect especially on acquired alopecia, preferably on alopecia without accompanying scarring or skin lesion, more preferably on alopecia areata, male pattern alopecia, seborrheic alopecia, alopecia pityroides, female pattern alopecia, gestational alopecia, malignant alopecia, senile alopecia, alopecia totalis, alopecia areata multilocularis, ophiasis, drug-induced alopecia, cancer chemotherapy-induced alopecia and radiation exposure-induced alopecia, traumatic/mechanical alopecia, malnutrition/metabolic disorder associated alopecia, endocrine dysfunction associated alopecia and telogen effluvium. Therefore, the external preparation of the present invention can be used for the purpose of treating or preventing at least one or more alopecia selected from the above-mentioned group.

One simple application onto aforementioned affected site at 3 μg/ml to 500 μg/ml improved or eliminated itch, dandruff, scalp redness and inflammation immediately thereafter (immediate effect). A single application stimulated hair growth, grew hair, provided hair with resilience, elasticity and increased volume, and dramatically decreased falling hairs, and these effects started on the day after the application and persisted for one week or more (long-lasting effect). If application was carried on for about one week, these effects are further improved, and even after terminating application, the effects lasted from 1 to 2 weeks such that stimulation of hair growth and restoration of hair continued and there were little or no falling hairs.

9.2 Confirming Effects on Skin

The obtained cyclic peptide was prepared in purified water at 3, 15, 30, 100 or 500 μg/ml and applied to affected site. Each of formulations A to E in the table indicates the concentration of the cyclic peptide at 3, 15, 30, 100 or 500 μg/ml, respectively. The absence of the effect upon the application of purified water without the peptide confirmed the effect was not a placebo effect.

TABLE 30 Amino acid sequence of cyclic peptide  Diagnostic Diagnostic impression on skin  Itches after  mutant used ID Sex Age impression Formulation Treatment after application application 1 SEQ ID NO 16: Q1  female 30′s eczema B once a day After 3 minutes, red papule was reduced  Itches were remitted  (CFVRKMDRISSSSGLGC) and remitted. immediately after  application. Q2  male 20′s atopic  B once a day After 2 minutes, erythema, edema and  Itches were eliminated dermatitis infiltration was remitted. immediately after  application. Q3  male 80′s plaque  C once a day, 2 After 3 minutes, crusts, scales, erythema psoriasis days and infiltration were remitted. Q4  male 40′s plaque  C once a day, After 10 minutes, scales, erythema and  psoriasis infiltration were remitted. Q5  female 30′s eczema C once a day After 3 minutes, erythema was remitted. Q6  male 20′s atopic  B once a day. 7 Erythema, infiltration and papule were  Itches were remitted  dermatitis days remitted. immediately after  application. Q7  female 60′s eczema C once a day After 2 minutes, erythema, infiltration  and papule were remitted. After 20 minutes, they were amaliorated. Q8  female 20′s atopic  C once a day After 2 minutes, erythema and  Itches were eliminated dermatitis infiltration were remitted. in 1 minute Q9  male 10′s atopic  C once a day Redness vanished immediately after  Itches were extincted  dermatitis application. in 1 minute. SEQ ID NO 17: Q10 male 40′s atopic  C once a day After 3 minutes, erythema and  After 3 minutes,  (CFGQKMDRISSSSGLGC) dermatitis infiltration were remitted. itches were remitted. Q11 male 10′s nummular C once a day After 3 minutes, infiltration, erythema  eczema exudate and scales were remitted. Q12 male 20′s eczema B once a day After 2 minutes, scales and erythema  Iches were elimintaed were remitted. With only 1 application, immediately after  no recurrence for 7 days thereafter. application. 2 SEQ ID NO 18: Q13 male 60′s milleria C once a day After 2 minutes, redness was remitted. (CFGQKMDRISSSSGLGC) Q14 female 40′s roughened A once a day After 2 minutes, zits, dryness and  skin rawness on skin were reduced, skin was  moisturized and it texture improved.  After 7 days, large wrinkles and  nasolabial fold became less deep. SEQ ID NO 19: Q15 male 20′s eczema, atopic C once a day After 2 minutes, erythema and scales were  (CFGRRMDRISSSSGLGC) dermatitis remitted. After 20 minutes, skin got  moisturized. After only 1 application,  erythema vanishes and kept in a good  condition for 1 week. Q16 male 20′s atopic  C once a day After 5 minutes, wound was apithelized,  dermatitis erythema and papule were improved. Q17 female 40′s roughened B once a day After 2 minutes, redness was remitted.  skin Roughness and skin texture were also  improved. Q18 female 30′s roughened B once a day After 2 minutes, was remitted. Roughness  skin and skin texture were also improved.  Rawness got better, iches vanished  immediately after application, skin  were moisturized and smooth. Q19 male 40′s plaque  C once a day After 3 minutes, erythema, crusts scales  psoriasis and infiltration were remitted. Q20 male 40′s eczema C once a day Erythema was remitted immediately after  Itches were extincted application. immediately after  application SEQ ID NO 20: Q21 female 60′s eczema C once a day After 1 minute, red papule was reduced  After 3 minutes,  (CFGRKLDRISSSSGLGC) and extincted. iches were remitted Q22 male 20′s atopic  B once a day After 2 minutes, erythema and scales  Itches vanished  dermatitis were remitted. completely immediately after application. Q23 female 40′s eczema C once a day After 2 minutes, erythema, scales and  Rawness vanished  papule were remitted. immediately after  Q24 female 30′s roughened B once a day After 3 minutes, zits, ruggedness, dry  Iches application were skin, acne skin and redness were remitted, skin got  eliminated immediately full and its texture improved. Acnes  after application. were reduced and redness was remitted. Q25 female 60′s wrinkles,  B once a day After 2 minutes, wrinkles became less  dry skin deep, dry skin was remitted. Q26 female 60′s eczema C once a day After 3 minutes, erythema and scales  were remitted. SEQ ID NO 21: Q27 male 20′s eczema, atopic C once a day After 3 days, erythema, scales and  Iches were eliminated (CFGQKIDRISSSSGLGC) dermatitis papule were improved. Cracks were  immediately after  epithelized, skin got moisturized and  application. improved. Q28 female 40′s eczema B once a day After 2 minutes, infiltration, erythema,  After 1 minute, iches papule and scratch wound were remitted. were eliminated. Q29 male 40′s atopic  B once a day Immediately after application, erythema After 3 minutes,  dermatitis and scales and remitted. iches were eliminated. 3 SEQ ID NO 22: Q30 female 30′s atopic  C once a day After 3 minutes, 1 application, erythema  After 30 seconds,  (CFGQKMDRISSSSGLGC) dermatitis and infiltration were remarkably remitted, Iches were eliminated. dry skin, scales and cracks were also  improved. Q31 female 70′s eczema C once a day immediately after application, erythema  Facial rawness  and papule remitted. vanished immediately  Q32 female 30′s atopic  B once a day, A remarkable improvement after 2 days,  after application. dermatitis 7 days and erythema was eliminated. Q33 female 40′s atopic  C once a day, After 1 minute, erythema was remitted. Iches were eliminated dermatitis 2 days After 2 minutes, scratch wound was  immediately after  epithelized. application After 3 minutes, skin texture was improved. Iches were eliminated With only 1-day applicatioin, erythema  immediately after  was eliminated, skin texture was  application improved with no dryness. Q34 male 20′s atopic  C once a day After 2 minutes, erythema and scales were Iches were remitted  dermatitis remitted. No recurrence for 7 days  immediately thereafter. after application Q35 female 50′s chronic eczema C once a day After 2 applications, lichenification  Iches were eliminated erythema was remitted. No recurrence  immediately after  for 3 weeks thereafter. application Q36 female 50′s roughened C once a day Skin roughness was cured immediately  skin after application. After 2 minutes, skin  got moisturized. Its redness faded. Skin  texture was finely improved. After 7  days, skin tone became brighter,spots  faded and flabiness was improved. Q37 female 30′s roughened B once a day After 2 minutes, zits, redness was  Rawness vanished  skin remitted. After 1 day, skin became  immediately after  normal tone and its texture was improved. application. SEQ ID NO 23: Q38 female 40′s atopic  C once a day After 2 minutes, scratch wound was  After 1 minute, acnes (CFGQKMDRISSSSGLGC) dermatitis epithelized and cured. Erythema was  were eliminated. improved. Q39 male 20′s eczema C once a day After 2 minutes, erythema, dry skin and  Itches were abeled  scales were improved and skin got  immediately after  moisturized. application Q40 male 20′s atopic  C once a day After 2 minutes, erythema, scales and  Itches were extincted  dermatitis infiltration were remitted. With 1  immediately after  application, no recurrence for 7 days. application. Q41 male 40′s plaque  C once a day After 3 minutes, crustsscaleserythema  psoriasis and infiltration were remitted. Q42 female 30′s roughened B once a day After 2 minutes, roughness, zits and  After 2 minutes,  skin redness were reduced, skin got moisturized itches were  and its texture was improved. extincted. Q43 female 50′s rosacea, acne C once a day After 1 minute, erythema, telangiectasia  and acne were remitted. After 2 minutes,  papule, infiltration, acne, dry skin and  redness was remitted. 4 SEQ ID NO 24: Q44 male 10′s atopic  C once a day After 3 minutes, erythema, scales and  (CFGQKMDRISASSGLGC) dermatitis infiltration were remitted. Further improvement after 40 minutes.  Scratch wound was epithelized. q45 male 20′s atopic  C once a day After 3 minutes, erythema and  After 3 minutes,  dermatitis infiltration were remitted. Iches were  Q46 male 20′s C once a day After 3 days, cracks were epithelized.  eliminated. Erythema, scales and infiltration were  remitted. Q47 male 20′s atopic  C once a day After 2 minutes, wound was epithelized.  Iches were eliminated  dermatitis Erythema, scales and licherification  immediately after  remitted. After 5 minutes, skin got  application. softened, erythema and infiltration were  improved. SEQ ID NO 25: Q48 male 20′s C once a day After 2 minutes, erythema,scales and  Iches were remitted  (CFGQKMDRISSQSGLGC) infiltration were remitted. Skin got  immediately after  moisturized. application. Q49 female 40′s roughened B once a day After 3 minutes, deep wrinkles were less  Rawness was eliminated  skin deep, dry skin and redness were improved, immediately after  skin got moisturized and full. By using  application. for 1 week, large wrinkles and nasolabial fold became less deep. Skin became  brighter. Spots faded.Flabiness was  improved. SEQ ID NO 26: Q50 male 60′s plaque  C once a day After 2 minutes, scales and infiltration  (CFGQKMDRISSSSGLGC) psoriasis remitted. Q51 male 30′s millaria C once a day After 2 minutes, remitted. Q52 male 20′s acne vulgaris C once a day After 6 minutes, acne became dry and was  reduced in size. Q53 female 60′s eczema C once a day After 3 minutes, erythema and infiltration were remitted. After 4 minutes, amelicrated. Q54 female 10′s atopic  C once a day After 2 minutes, strong itches almost  dermatitis vanished. Erythema and lichenification  were improved. Skin got softened. Q55 female 40′s roughened C once a day After 2 minutes, zits, redness and  Rawness vanished  skin roughness were improved. Skin got  immediately after  moisturized and its texture was  application. improved. Skin was improved. SEQ ID NO 27: Q56 female 50′s roughened C once a day After 3 minutes, dry skin was improved,  (CFGQKMDRISSSSGLGC) skin, acne skin got moisturized and its texture was  finely improved. Redness vanished.  Inframmation in acne was remitted,  redness faded and dried. By using for 1  week, skin became brighter. Q57 male 20′s atopic  C once a day After 3 minutes, erythema, infiltration Iches were eliminated dermatitis and lichenification was improved. immediately after  application. 5 SEQ ID NO 28: Q58 female 30′s eczema C once a day After 1 minute, erythema and infiltration  After 1 minute,  (CFGQKMDRISSISGLGC) were remitted. Iches were  Q59 female 20′s roughened A once a day After 7-day continuous application, skin  eliminated. skin roughness vanished, wrinkles were less  deep, and skin became brighter. Q60 female 20′s roughened C once a day After 2 minutes, scales were improved.  skin With 1 application/day, roughness was  improved and skin could be kept  moisturized. SEQ ID NO 29: Q61 female 30′s eczema C once a day After 1 minute, erythema and  Iches were eliminated (CFGQKMDRISSMSGLGC) infiltration were remitted. immediately after  application. SEQ ID NO 30: Q62 male 20′s atopic  B once a day, After 3 days, erythema and infiltration (CFGQKMDRISSSVGLGC) dermatitis 7 days were remitted. Q63 female 60′s eczema C once a day After 1 minute, erythema and papule were  Iches were eliminated remitted. After 3 days, there were  immediately after  almost cured. application. Q64 male 50′s dry skin C once a day Immediately after application, skin got  promptly moisturized and its texture was  improved, zits, erythema and roughned  skin were improved. Q65 male 20′s atopic  C once a day After 10 minutes, lichenification was  Iches were eliminated dermatitis improved. Skin got softened, erythema  immediately after  and scales were improved. application. Q66 female 40′s roughened A once a day After 1 day with 1 application, skin  skin conditions of roughness, zits and itches  were improved and skin texture was finely  improved. The effect lasted for 7 days  thereafter. SEQ ID NO 31: Q67 male 20′s atopic  C once a day After 2 minutes, erythema and papule  (CFGQKMDRISSSSRLGC) dermatitis was remitted. SEQ ID NO 32: Q68 male 30′s millaria C once a day After 2 minutes, red papule was reduced  (CFGQKMDRISSSSGMGC) in size, skin dryness was remitted. Q69 male 10′s atopic  C once a day After 3 minutes, exudate stopped,  dermatitis, erythema and infiltration were improved. numeuler 6 SEQ ID NO 33: Q70 female 30′s contact C 2 applicatioin After 3 minuts, erythema, infiltration  After 1 minute, iches (CFGQKMDRISSSSGIGC) dermatits and eczema were remitted. were eliminated. Q71 female 30′s atopic  E once a day Immediately after application, erythema  Iches were eliminated dermatitis and scales were improved. immediately after  Q72 female 70′s plaque  C 2 application After 5 minutes. erythema, scales  application. psoriasis and infiltration were remitted. Q73 female 40′s roughened B once a day In 2 minutes after application, skin  skin was improved. Skin texture was finely  improved. Skin got softened. SEQ ID NO 34: Q74 female 30′s eczema C 2 applicatioin After 3 minutes, erythema, infiltration  After 1 minute, Iches (CFGQKMDRISSSSGVGC) and edema remitted. were eliminated. Q75 male 20′s eczema, acne C 2 applicatioin After 5 minutes, acne was dried and  vulgaris reduced in size. Erythema and scales  were remitted. Q76 female 70′s plaque  C 2 applicatioin After 5 minutes, erythema, scales and  psoriasis infiltration were remitted. Q77 female 40′s roughened C once a day After 2 minutes, skin texture was  skin improved, skin got hull, and redness  and dryness were improved. Q78 male 50′s roughened B once a day After 3 minutes, skin got moisturized  skin and its texture was improved. Q79 female 20′s atopic  C once a day After 3 minutes, erythema and scales  Itches were improved  dermatitis were improved. immediately after  applilcation. Q80 female 50′s roughened B once a day After 3 minutes, skin roughness was  Iches wee elminated  skin improved, skin got moisturized and its  immediately after  texture was fimely improved. Skin gained  application. resilence .After 7 days, large wrinkles  and nasolabial fold because less deep.  By continuing 4 weeks, spots faded and  skin tone became brighter. Skin resilence was improved. Large wrinkles became less  deep. Q81 male 30′s roughened B once a day After 3 minutes, skin roughness and  skin redness were improved. Skin texture  was finely improved. By continuing for  7 days, skin resilence was improved. SEQ ID NO 35: Q82 female 40′s roughened B once a day After 3 minutes, roughness skin and dry  (CFGQKMDRISSSSGAGC) skin skin were promptly improved, skin  texture was improved. Q83 male 60′s plaque  C twice a day After 30 minutes, erythema, scales and  psoriasis infiltration were improved. Q84 male 40′s plaque  C twice a day After 20 minutes, erythema and scales  psoriasis were improved. Q85 female 70′s plaque  C twice a day After5 minutes, erythema and scales  psoriasis were remitted. 7 SEQ ID NO 36: Q86 female 10′s atopic  C once a day After 2 minutes, linchenification was  After 2 minutes,  (CFGQKMDRISSSSGLSC) dermatitis remarkably remitted, erythema, scales  strong iches were  inflimation, cracks, scratch wound,  eliminated. etc. all remitted. After 5 minutes,  improved with only mild infiltration  left. Q87 female 20′s atopic  B once a day After 1 minute, erythema, infiltration  After 2 minutes,  dermatitis and papule remitted. After 4 minutes,  iches were  infiltration was remitted with only  eliminated. mild erythema left. Q88 male 10′s atopic  C once a day After 3 minutes, erythema, scales,  After 3 minutes,  dermatitis infiltration and scratch wound were  iches were  remitted. eliminated. Q89 female 40′s roughened B once a day Immediately after application, itches  skin and redness were improved. After 3  minutes, skin got full and resilent,  and skin was improved. Q90 male 50′s seborrhea C once a day, Immediately after application,seborrhea  Itches were remitted dermatits 3 days was remitted. After 3 days, erythema  immediately after  and seborrhea was remiited. application. SEQ ID NO 37: Q91 male 60′s plaque  C once a day After 2 minutes, erythema and scales  (CFGQKMDRISSSSGLAC) psoriasis were remitted. Q92 male 20′s atopic  C once a day After 2 minutes, erythema and  Iches were eliminated  dermatitis infiltration were remitted. Applied  immediately after  prurigo twice. After 15 minutes, pringe nodularis  application. nodularis, on hip was remitted Q93 female 40′s roughened B once a day After 3 minutes, roughened skin and dry  skin skin were improved promptly and skin  texture was improved. Q94 male 40′s plaque  C once a day After 20 minutes, erythema and scales  psoriasis were remitted. Q95 male 30′s atopic  C once a day After 2 minutes, scratch wound  dermatitis epithelized erythema was remitted. SEQ ID NO 38: Q96 male 20′s atopic  C once a day After 3 minutes, cracks was reduced in  immediately after  (CFGQKMDRISSSSGLGC) dermatitis size. Erythema, scales and  application, itches lichenification were remitted. and rawness were  removed. Q97 female 40′s roughened B once a day After 1 minute, dry skin was improved,  immediately after  skin skin got moiturized and full, and  application, itches  skin texture was improved. were removed. Q98 female 200′s chronic C once a day immediately after application, erythema  eczema and scales were remitted. Q99 male 20′s atopic  C once a day After 2 minutes, redness was improved.  After 1 minute, iches dermatitis After 3 minutes, erythema, infiltration  were eliminated.  and papule were remitted. After 1  After 1 application, application, the effect lasted for 6  no itches for 6 days  days with no recurrence. thereafter. Q100 female 50′s prurigo C once a day After 3 minutes, nodes wereflatened,  Iches were eliminated  nodularis, scratch wound became dry and epithelized  immediately after  eczema with no more exudate. application. 8 SEQ ID NO 39: Q101 female 50′s prurigo C once a day After 3 minutes, nodes were flattened. (CFSRKMDRISSSSGLGC) nodularis, eczema Q102 female 10′s chronic eczema C once a day After 2 minutes, an intractable eczema  that cannot be remitted by applying strong steriod ointments was remitted, and  erythema, scales and papule were reduced. Q103 female 50′s eczema C once a day After 2 minutes, erythema, scales and  pigmentation were remitted. Skin got full  and soft, with fresh color. Q104 female 20′s atopic  D once a day Scratch wound was epithelized Exudate was  Iches were eliminated dermatitis dried ad lichenification was remitted.  immediately after  erythema and infiltration were remitted. application. Q105 male 20′s atopic  B once a day After 3 minutes, erythema and infiltration After 2 minutes,  dermatitis were remitted. With 1 application, the  itches and tingling  effect persisted for 1 week with no  pain were removed.   recurrence. The effect persisted for 7 days with no  itches. SEQ ID NO 40: Q106 male 40′s atopic  C once a day After 2 minutes, erythema, infiltration  Iches were eliminated (CFTRKMDRISSSSGLGC) dermatitis and lichenification remitted. immediately after  application. Q107 female 20′s roughened C 1 application After 1 minute, dry skin was improved,  lips cracks were less deep and apithelized.  Q108 female 40′s roughened B once a day After 3 minutes, redness and roughness  skin were improved. Q109 female 30′s healthy skin C once a day After 3 minutes, dark-reddish skin tone  was improved to turn fresh color.  Dryness, tautnessm rawness and hot flash  of skin were removed. With 1 application the effect persisted for 1 week. 9 SEQ ID NO 41: Q110 male 10′s atopic  C once a day immediately after application, erythema  Iches were eliminated  (CFGRKMDRISSTSGLGC) dermatitis/ and infiltration of eczema were remitted.  immediately after  application. acne The effects were exhibited remarkably  fast. After 3 minutes, erythema was  further remitted. Acne was dried and  reduced in size. Q111 male 20′s atopic  C once a day Immediately after application, erythema  Iches were eliminated  dermatitis and infiltration were remitted. After  immediately after  20 minutes, deep scratch wound was  application. epithelized. Q112 female 40′s roughened B 1 application After 1 minute, skintautness and rawness  lips were improved and skin got moisturized. Q113 female 50′s atopic  B once a day Iches stopped in 30 seconds. Redness  dermatitis and edema were remitted in 1 minute. Q114 female 10′s roughened B once a day After 1 minute, redness was remitted. After 3 minutes, skin skin/acne After 3 minutes, skin roughness, zits,  tautness was removed. rough and soft skin texture were improved,  skin got full end its texture was finely improved. Acne became dry and reduced in  size. Q115 female 40′s roughened B once a day Rawness vanished in 1 minute. Skin was  skin moisturized. After 2 minutes, redness,  roughness and stiffness were remarkably  improved. By using for 1 weel, skin  became brighter, flabbiness was improved. SEQ ID NO 42: Q116 female 40′s acne C once a day After 1 minute, redness was remitted. (CFGRKMDRISSASGLGC) Q117 male 10′s atopic  B once a day After 3 minutes, erythema and  Iches were eliminated dermatitis infiltration were remitted. immediately after  application. Q118 female 50′s eczema C once a day After 3 minutes, erythema and dry skin  remitted. SEQ ID NO 43: Q119 male 40′s atopic  C once a day After 2 minutes, no discomfort, felt  After 3 minutes,  (CFGRKMDRISSSTGLGC) dermatitis fresh. Erythema, infiltration and  roughness and  lichenification were remitted. stiffness were  removed, and itches  vanished. Q120 female 30′s roughened C once a day 1 minute after application, dry skin  lips was improved cracks became less deep  and epithelized. Q121 female 40′s roughened B once a day After 3 minutes, redness and dry skin  skin were improved. Skin got moisturized  promptly, and skin texture was improved.  Skin rawness, stiffness and irritation  were eliminated. Pores became less  distinguised. Q122 female 40′s rosacae/acne C once a day After 7 minutes, acne was reduce and  became dry. Q123 female 30′s eczema C once a day After 1 minute, redness and infiltration  Iches were eliminated  were remitted. immediately after  application. 10 SEQ ID NO 44: Q124 female 60′s healthy skin B once a day Skin got moisturized promptly, wrinkles  (CFGRKMDRISSSAGLGC) became less deep, skin texture was improved. By using for 4 weeks, large wrinkles and  nasolabial fold became less deep. Skin  resilence and flabiness were improved.  Spots faded. Skin tone became brighter. Q125 female 80′s eczema C once a day After 1 minute, erythema and infiltration  Iches were remitted  were remitted. immediately after  application. SEQ ID NO 45: Q126 female 40′s roughened B once a day After 2 minutes, dryness, tautness and  (CFSRRMDRISSSSGLGC) skin rawness of skin were eliminated, skin got  full, and wrinkles became less deep. Skin  mained elasticity. By using for 5 days,  flabbiness was improved. Skin became  brighter, nasolabial fold became less  deep. By using for 4 weeks, nasolabial  fold became less deep. Skin resilence  and flabbiness were improved. Spots faded  and skin tone became brighter. SEQ ID NO 46: Q127 female 40′s eczema C once a day After 2 minutes, erythema, scales and  (CFGRKMDRISSTSGLGC) infiltration were remitted. Skin got  softened. SEQ ID NO 47: Q128 female 30′s chronic eczema C once a day Immediately after application, erythema  (CFGRKMDRISSSTGLGC) and scales were remitted. SEQ ID NO 48: Q129 male 50′s eczema C once a day After 2 minutes, erythema, scales and  (CFGRKMDRISSSSGIGC) infiltration were remitted. Skin got  moisturized. SEQ ID NO 49: Q130 male 30′s chronic eczema C once a day Immediately after application, erythema  (CFGRKMDRISSSSGLAC) and scales were remitted. SEQ ID NO 50: Q131 male 50′s eczema C once a day After 2 minutes, erythema, scales and  (CFGRRMDRISSSTGLGC) infiltration were remitted. SEQ ID NO 51: Q132 female 60′s eczema B once a day After 3 minutes, erythema and  (CFGRRMDRISSSTGLGC) infiltration were remitted. SEQ ID NO 52: Q133 female 50′s eczema C once a day After 2 minutes, erythema, scales and  (CFGRRMDRISSSSGIGC) infiltration were remitted. Skin got  moisturized. SEQ ID NO 53: Q134 female 20′s eczema C once a day After 1 minute, erythema and infiltration  (CFGRRMDRISSSSGLAC) were remitted. SEQ ID NO 54: Q135 female 30′s roughened B once a day After 2 minutes, dryness, tautness and  (CFSRKMDRISSTSGIGC) skin rawness of skin were eliminated, skin  got full and wrinkles became less deep.  Skin gained elasticity. SEQ ID NO 54: Q136 female 50′s eczema C once a day After 2 minutes, erythema, scale and  (CFSRKMDRISSTSGIGC) infiltration were remitted. 11 SEQ ID NO 56: Q137 male 20′s roughened B once a day After 2 minutes, dryness, tautness and  (CFGRKMDRISSSTGIGC) skin rawness of skin were elimnated, skin got  full, and wrinkles became less deep.  Skin gained elasticity. SEQ ID NO 57: Q138 female 40′s eczema C once a day After 2 minutes, erythema, scales and  (CFGRKMDRISSSTGLAC) infiltration were remitted, scratch  wound was epithelized and dried. SEQ ID NO 58: Q139 female 30′s eczema C once a day After 2 minutes, erythema, scales,  (CFGRKMDRISSSSGIAC) papule and infiltration were remitted. SEQ ID NO 59: Q140 male 60′s roughened B once a day After 2 minutes, dryness, tautness and  (CFGRKMDRISSTSGLGC) skin rawness of skin were eliminated. Skin  gained elasticity, wrinkles became less  deep. SEQ ID NO 60: Q141 female 50′s chronic C once a day Immediately after application, erythema  (CFGRRMDRISSSTGLGC) eczema and scales were remitted. SEQ ID NO 61: Q142 female 30′s chronic C once a day Immediately after application, erythema  (CFSRRMDRISSSSGIGC) and scales were remitted. SEQ ID NO 62: Q143 female 40′s roughened B once a day After 2 minutes, dryness, tautness and  (CFSRRMDRISSSSGLAC) skin rawness of skin were eliminated, skin  got full, and wrinkles became less deep.  Skin gained elasticity. SEQ ID NO 63: Q144 male 50′s eczema B once a day After 3 minutes, erythema and scales  (CFSRKMDRISSTSGIGC) infiltration were remitted, and skin got  softened. SEQ ID NO 64: Q145 male 40′s chronic C once a day Immediately after application, erythema  (CFSRKMDRISSTSGIGC) eczema and scales were remitted. SEQ ID NO 65: Q146 female 40′s roughened C once a day After 1 minute, skin got moisturized and  (CFGRKMDRISSSTGLAC) skin resilent, skin texture was improved. SEQ ID NO 66: Q147 male 50′s eczema C once a day After 2 minutes, erythema, scales and  (CFGRKMDRISSSSGIAC) infiltration were remitted. SEQ ID NO 67: Q148 female 70′s roughened B once a day After 2 minutes, dryness, tautness and  (CFSRRMDRISSTSGIGC) skin rawness of skin were eliminated, skin  got full, and wrinkles became less deep.  Skin gained elasticity. SEQ ID NO 68: Q149 female 30′s chronic C once a day Immediately after application, erythema  (CFGRRMDRISSTSGLAC) eczema and scales were remitted. SEQ ID NO 69: Q150 female 20′s eczema C once a day After 2 minutes, erythema, scales,  (CFSRRMDRISSTSGIGC) infiltration were improved. 12 SEQ ID NO 70: 151 female 60′s chronic C once a day immediately after application, erythema  (CFGRRMDRISSSTGLAC) eczema and scales were remitted. 152 female 30′s roughened C once a day Redness vanished in 30 seconds. After 1  skin minute, skin texture improved finely.  SEQ ID NO 71: Pores became less distinguished. (CFSRRMDRISSTSGIGC) 153 male 50′s roughened A once a day After 2 minutes, dryness, tautness and  skin rawness of skin were eliminated, skin  got full and wrinkles became less deep.  Skin gained elasticity. SEQ ID NO 72: 154 male 50′s chronic C once a day Immediately after application, erythema,  (CFSRRMDRISSTSGLAC) eczema scales and papule were remitted. SEQ ID NO 73: 155 male 20′s chronic C once a day Immdediately after application, erythema  (CFSRRMDRISSSTGIGC) eczema and scales were remitted. SEQ ID NO 74: 156 female 30′s roughened C once a day After 1 minute, rawness and hot flash  (CFGRRMDRISSSTGLAC) skin were eliminated, skin got moisturized  and its texture improved, wrinkles faded  and skin gained elasticity. 157 male 60′s chronic D once a day Iches were eliminated immediately after  eczema application. Redness was remitted. SEQ ID NO 75: 158 female 60′s eczema C once a day After 2 minutes, erythema, scalesm  (CFGRRMDRISSSSGIAC) papule, infiltration were remitted. SEQ ID NO 43: 258 male 40′s atopic  C once a day After 2 minutes, cracks were epithelized  (CFGRKMDRISSSTGLGC) dermatitis with no more pains and itches. Erythema  and and scales were also remitted. SEQ ID NO 19: (CFGRRMDRISSSSLGC) SEQ ID NO 43: 358 male 40′s eczema once a day After 2 minutes, wound was epithelized  (CFGRKMDRISSSTGLGC) quickly, and erythema, scales and  and infiltration were remarkably remitted. SEQ ID NO 99: In SEQ ID NOS 16-75, 1st Cys and 17th Cys from left form a disulfide bond.

The result described above demonstrated that the cyclic peptide of the invention exhibits a similar effect as the cyclic peptide expressed by Formula I-a even if some amino acids of the cyclic peptide have been replaced. Moreover, a similar effect could be obtained by applying the replaced cyclic peptide in a mixture as long as an effective amount of the cyclic peptide is applied.

Specifically, in subjects having atopic dermatitis or eczema, effects were observed immediately after applying 3 μg/ml to 500 μg/ml to affected site, including elimination of itches, epithelialization at infiltrated site, papule or scratch, termination of exudation, epithelialization of a crack. Moreover, a single application induced a persistent effect for a week or more.

When it was applied to healthy or roughened skin, it exerted a moisturizing effect immediately or within a few minutes after application, improving dryness, providing and keeping skin and mucosa with moderate elasticity and flexibility, softening skin, giving skin or mucosa resilience and firmness. Also, fine wrinkles and flabbiness are improved at the applied site, and dullness and spots are further faded. A single application induced a persistent effect for a week or more.

9.3 Confirming Nasal Effect

The obtained cyclic peptide was prepared in physiological saline at 3, 15, 30, 100 or 500 μg/ml, and 0.1 ml each was applied to nasal cavity. Each of formulations A to E in the table indicates the concentration of the cyclic peptide at 3, 15, 30, 100 or 500 μg/ml, respectively. The absence of the effect upon the application of physiological saline without the peptide confirmed the effect was not a placebo effect.

TABLE 13-1 SEQ ID NO (amino acid sequence) of  Diagnostic Formu- Diagnostic impression on Itches cyclic peptides ID Sex Age impression lation Treatment skin after application after application SEQ ID NO 16: P1 female 10's allergic C once a day, 0.1 ml From immediately after nasal Itches were remitted (CFVRKMDRISSS rhinitis applied to each application, and nose got immediately after nasal SGLGC) nasal cavity cleared, rhinorrhea stopped. application, and eliminated There was no irritationg after 1 minute. symptoms, and the effect persisted for all day by 1 nasal application. SEQ ID NO 17: P2 male 30's allergic D once a day, 0.1 ml From immediately after nasal Itches were remitted (CFGQKMDRISSS rhinitis applied to each application, and nose got immediately after nasal SGLGC) nasal cavity cleared, rhinorrhea stopped. application, and eliminated There was no irritationg after 1 minute. symptoms, and the effect persisted for all day by 1 nasal application. SEQ ID NO 18: P3 male 50's allergic C once a day, 0.1 ml After 3 minutes, and nose (CFGHKMDRISSS rhinitis applied to each got cleared, rhinorrhea SGLGC) nasal cavity stopped. There was no irritationg symptoms, and the effect persisted for all day by 1 nasal application. SEQ ID NO 19: P4 female 40's chronic B once a day, 0.1 ml After 1 minute, and nose got (CFGRRMDRISSS rhinitis, applied to each cleared, nasal discharge SGLGC) perennial nasal cavity stopped. There was no rhinitis irritating symptoms. P5 female 70's allergic B once a day, 0.1 ml After 2 minutes, and nose Itches were remitted rhinitis applied to each got cleared, and no nasal immediately after nasal nasal cavity discharge came out by application. blowing nose. SEQ ID NO 20: P6 female 40's allergic B once a day, 0.1 ml After 1 minute, nasal (CFGRKLDRISSS rhinitis applied to each obstruction was improved, SGLGC) nasal cavity and nose got cleared. The effect persisted for all day. SEQ ID NO 21: P7 female 20's allergic C once a day, 0.1 ml After 1 minute, and nose got (CFGRKIDRISSS rhinitis applied to each cleared. Nasal discharge SGLGC) nasal cavity stopped. SEQ ID NO 22: P8 female 50's allergic E once a day, 0.1 ml Immediately after nasal Immediately after nasal (CFGRKMDRVSSS rhinitis applied to each application, and nose got application, restless itches SGLGC) nasal cavity cleared. and sneezing stopped. P9 female 40's chronic C once a day, 0.1 ml After 20 seconds, and nose rhinitis, applied to each got cleared. No nasal perennial nasal cavity discharge after 1 minute. rhinitis There was no irritating symptoms. SEQ ID NO 23: P10 female 30's allergic C once a day, 0.1 ml After 1 minute, and nose got (CFGRKMDRIGSS rhinitis applied to each cleared. Nasal discharge SGLGC) nasal cavity stopped. SEQ ID NO 24: P11 female 20's allergic C once a day, 0.1 ml After 1 minute, and nose got (CFGRKMDRISAS rhinitis applied to each cleared. Nasal discharge SGLGC) nasal cavity stopped. SEQ ID NO 25: P12 female 20's allergic A once a day, 0.1 ml After 2.5 minutes, and nose (CFGRKMDRISSQ rhinitis applied to each got cleared. SGLGC) nasal cavity SEQ ID NO 26: P13 female 50's allergic C once a day, 0.1 ml After 1 minute, nose got (CFGRKMDRISSV rhinitis applied to each cleared and refreshed. After SGLGC) nasal cavity 2 minutes, no nasal discharge by blowing. SEQ ID NO 27: P14 male 20's allergic C once a day, 0.1 ml After 1 minute, and nose got (CFGRKMDRISSL rhinitis applied to each cleared. SGLGC) nasal SEQ ID NO 28: P15 female 40's allergic C once a day, 0.1 ml After 1 minute, and nose got (CFGRKMDRISSI rhinitis applied to each cleared. Nasal discharge SGLGC) nasal cavity stopped. SEQ ID NO 29: P16 male 50's allergic C once a day, 0.1 ml After 1 minute, and nose got (CFGRKMDRISSM rhinitis applied to each cleared. Nasal discharge SGLGC) nasal cavity stopped. SEQ ID NO 30: P17 female 30's allergic C once a day, 0.1 ml After 1 minute, rhinorrhea (CFGRKMDRISSS rhinitis applied to each stopped. VGLGC) nasal cavity SEQ ID NO 31: P18 female 10's allergic D once a day, 0.1 ml After 30 seconds, rhinorrhea (CFGRKMDRISSS rhinitis applied to each stopped. After 1 minute, no SRLGC) nasal cavity nasal discharge by blowing. SEQ ID NO 32: P19 male 40's allergic C once a day, 0.1 ml After 1 minute, nasal (CFGRKMDRISSS rhinitis applied to each obstruction was improved, SGMGC) nasal cavity and nose got cleared. The effect persisted for all day. P20 female 40's chronic C once a day, 0.1 ml Immediately after nasal rhinitis, applied to each application nose got perennial nasal cavity cleared. The effect was rhinitis remarkable and persisted for 7 days after 1 nasal application. SEQ ID NO 33: P21 female 40's allergic C once a day, 0.1 ml After 30 seconds, rhinorrhea Sneezing and itches in deep (CFGRKMDRISSS rhinitis applied to each stopped. After 1 minute, no inside nose stopped. SGIGC) nasal cavity nasal discharge by blowing. SEQ ID NO 34: P22 female 40's allergic C once a day, 0.1 ml After 2.5 minutes, and nose No irritating symptoms. (CFGRKMDRISSS rhinitis applied to each got cleared. SGVGC) nasal SEQ ID NO 35: P23 female 30's allergic C once a day, 0.1 ml After 1 minute, nasal (GFGRKMDRISSS rhinitis applied to each obstruction was improved, SGAGC) nasal cavity and nose got cleared. The effect persisted for all day. P24 female 40's chronic D once a day, 0.1 ml Immediately after nasal rhinitis, applied to each application, nose got perennial nasal cavity refreshed and cleared. rhinitis SEQ ID NO 36: P25 female 40's allergic C once a day, 0.1 ml Immediately after nasal Immediately after nasal (CFGRKMDRISSS rhinitis applied to each application, and nose got application, itches were SGLSC) nasal cavity cleared. removed. No irritating symptoms. SEQ ID NO 37: P26 female 40's allergic C once a day, 0.1 ml After 30 seconds, rhinorrhea Immediate effect to remove (CFGRKMDRISSS rhinitis applied to each stopped. Nose got cleared itches. SGLAC) nasal cavity and felt good. Other nose drops causes this subject to sneeze due to some irritant, but the inventive nose drop had no irritation. The effect persisted for 1 week by 1 nasal application. P27 female 40's chronic C once a day, 0.1 ml Nose got cleared and rhinitis, applied to each rhinorrhea stopped in 30 perennial nasal cavity seconds. rhinitis SEQ ID NO 38: P28 male 30's chronic C once a day, 0.1 ml After 2 minutes, rhinorrhea (CFARKMDRISSS rhinitis applied to each stopped. Nasal obstruction SGLGC) nasal cavity was improved to allow nasal breathing. SEQ ID NO 39: P29 male 40's allergic C once a day, 0.1 ml Immediately after nasal (CFSRKMDRISSS rhinitis applied to each application, nose got SGLGC) nasal cavity refreshed and cleared. No nasal discharge after 30 seconds. SEQ ID NO 40: P30 female 30's allergic C once a day, 0.1 ml After 2.5 minutes, and nose (CFTRKMDRISSS rhinitis applied to each got cleared. SGLGC) nasal SEQ ID NO 41: P31 female 20's allergic C once a day, 0.1 ml After 1 minute, rhinorrhea (CFGRKMDRISST rhinitis applied to each stopped. There was no SGLGC) nasal cavity irritating symptoms. SEQ ID NO 42: P32 female 10's allergic C once a day, 0.1 ml Immediately after nasal (CFGRKMDRISSA rhinitis applied to each application, and nose got SGLGC) nasal cavity cleared. SEQ ID NO 43: P33 male 20's allergic C once a day, 0.1 ml Immediately after nasal (CFGRKMDRISSS rhinitis applied to each application, and nose got TGLGC) nasal cavity cleared. After 1 minute, no nasal discharge even by twisting nose. SEQ ID NO 44: P34 male 40's allergic C once a day, 0.1 ml After 1 minute, rhinorrhea (CFGRKMDRISSS rhinitis applied to each stopped. There was no AGLGC) nasal cavity irritating symptoms. SEQ ID NO 45: P35 male 60's allergic C once a day, 0.1 ml After 1 minute, and nose got (CFSRRMDRISSS rhinitis applied to each cleared. SGLGC) nasal SEQ ID NO 47: P36 male 50's allergic C once a day, 0.1 ml After 1 minute, rhinorrhea (CFSRKMDRISSS rhinitis applied to each stopped. There was no TGLGC) nasal cavity irritating symptoms. SEQ ID NO 48: P37 female 50's allergic C once a day, 0.1 ml After 1 minute, and nose got (CFSRKMDRISSS rhinitis applied to each cleared. Nasal discharge SGIGC) nasal cavity stopped. SEQ ID NO 49: P38 female 40's allergic C once a day, 0.1 ml Immediately after nasal (CFSRKMDRISSS rhinitis applied to each application, and nose got SGLAC) nasal cavity cleared. SEQ ID NO 50: P39 female 10's allergic C once a day, 0.1 ml Immediately after nasal (CFGRRMDRISST rhinitis applied to each application, and nose got SGLGC) nasal cavity cleared. SEQ ID NO 51: P40 male 20's chronic C once a day, 0.1 ml After 1.5 minutes, nasal (CFGRRMDRISSS rhinitis applied to each obstruction was improved, TGLGC) nasal cavity and nose got cleared. SEQ ID NO 52: P41 male 30's allergic C once a day, 0.1 ml After 1 minute, rhinorrhea (CFGRRMDRISSS rhinitis applied to each stopped. There was no SGIGC) nasal cavity irritating symptoms. SEQ ID NO 53: P42 male 40's allergic C once a day, 0.1 ml Immediately after nasal (CFGRRMDRISSS rhinitis applied to each application, and nose got SGLAC) nasal cavity cleared. SEQ ID NO 54: P43 female 30's allergic C once a day, 0.1 ml Immediately after nasal (CFGRKMDRISST rhinitis applied to each application, and nose got SGIGC) nasal cavity cleared. SEQ ID NO 55: P44 male 50's allergic C once a day, 0.1 ml After 1 minute, and nose got (CFGRKMDRISST rhinitis applied to each cleared. Nasal discharge SGLAC) nasal cavity stopped. SEQ ID NO 56: P45 male 30's allergic C once a day, 0.1 ml Immediately after nasal (CFGRKMDRISSS rhinitis applied to each application, and nose got TGIGC) nasal cavity cleared. SEQ ID NO 57: P46 female 40's allergic C once a day, 0.1 ml After 1 minute, and nose got (CFGRKMDRISSS rhinitis applied to each cleared. Nasal discharge TGLAC) nasal cavity stopped. SEQ ID NO 58: P47 female 40's allergic C once a day, 0.1 ml After 30 seconds, rhinorrhea Itches were removed in (CFGRKMDRISSS rhinitis applied to each stopped, with no more 30 seconds. SGIAC) nasal cavity sneezing. There was no irritating symptoms. SEQ ID NO 59: P48 female 20's allergic C once a day, 0.1 ml Immediately after nasal (CFSRRMDRISST rhinitis applied to each application, and nose got SGLGC) nasal cavity cleared. SEQ ID NO 60: P49 female 40's allergic C once a day, 0.1 ml After 1 minute, and nose got (CFSRRMDRISSS rhinitis applied to each cleared. Nasal discharge TGLGC) nasal cavity stopped. SEQ ID NO 61: P50 male 30's allergic C once a day, 0.1 ml Immediately after nasal (CFSRRMDRISSS rhinitis applied to each application, and nose got SGIGC) nasal cavity cleared. SEQ ID NO 62: P51 female 40's allergic C once a day, 0.1 ml After 1 minute, rhinorrhea (CFSRRMDRISSS rhinitis applied to each stopped, with no more SGLAC) nasal cavity sneezing. There was no irritating symptoms. SEQ ID NO 63: P52 female 30's allergic C once a day, 0.1 ml Immediately after nasal (CFSRKMDRISST rhinitis applied to each application, and nose got SGIGC) nasal cavity cleared. SEQ ID NO 64: P53 male 60's allergic C once a day, 0.1 ml After 1 minute, rhinorrhea (CFSRKMDRISSS rhinitis applied to each stopped, with no more TGIGC) nasal cavity sneezing. There was no irritating symptoms. SEQ ID NO 65: P54 female 30's allergic C once a day, 0.1 ml After 1 minute, and nose got (CFSRKMDRISSS rhinitis applied to each cleared. Nasal discharge TGLAC) nasal cavity stopped. SEQ ID NO 66: P55 female 30's allergic C once a day, 0.1 ml Immediately after nasal (CFSRKMDRISSS rhinitis applied to each application, and nose got SGIAC) nasal cavity cleared. SEQ ID NO 67: P56 female 40's allergic C once a day, 0.1 ml After 1 minute, and nose (CFGRRMDRISST rhinitis applied to each got cleared. Nasal SGIGC) nasal cavity discharge stopped. SEQ ID NO 68: P57 male 60's allergic C once a day, 0.1 ml After 1 minute, rhinorrhea (CFGRRMDRISST rhinitis applied to each stopped, with no more SGLAC) nasal cavity sneezing. There was no irritating symptoms. SEQ ID NO 69: P58 male 20's allergic C once a day, 0.1 ml Immediately after nasal (CFGRRMDRISSS rhinitis applied to each application, and nose got TGIGC) nasal cavity cleared. SEQ ID NO 70: P59 female 40's allergic C once a day, 0.1 ml Immediately after nasal (CFGRRMDRISSS rhinitis applied to each application, and nose got TGLAC) nasal cavity cleared. SEQ ID NO 71: P60 female 30's allergic C once a day, 0.1 ml After 1 minute, rhinorrhea (CFSRRMDRISST rhinitis applied to each stopped, with no more SGIGC) nasal cavity sneezing. There was no irritating symptoms. SEQ ID NO 72: P61 female 40's allergic C once a day, 0.1 ml After 1 minute, rhinorrhea (CFSRRMDRISST rhinitis applied to each stopped, with no more SGLAC) nasal cavity sneezing. There was no irritating symptoms. SEQ ID NO 73: P62 female 20's allergic C once a day, 0.1 ml Immediately after nasal (CFSRRMDRISSS rhinitis applied to each application, and nose got TGIGC) nasal cavity cleared. SEQ ID NO 74: P63 female 40's allergic C once a day, 0.1 ml After 1 minute, rhinorrhea (CFSRRMDRISSS rhinitis applied to each stopped, with no more TGLAC) nasal cavity sneezing. There was no irritating symptoms. SEQ ID NO 75: P64 male 20's allergic C once a day, 0.1 ml After 1 minute, and nose (CFSRRMDRISSS rhinitis applied to each got cleared. Nasal SGIAC) nasal cavity discharge stopped. In SEQ ID NOS 16-75, 1st Cys and 17th Cys from left form a disulfide bond.

The result described above demonstrated that the cyclic peptide of the invention exhibits a similar effect as the cyclic peptide expressed by Formula I-a even if some amino acids of the cyclic peptide have been replaced.

Specifically, when it was applied to nasal cavity mucosa and/or paranasal cavity mucosa, it can improve or prevent various symptoms associated with rhinitis such as nasal obstruction, rhinorrhea, sneezing and pruritus. Rhinitis also includes, without being particularly limited, for example, infectious rhinitis including acute rhinitis and chronic rhinitis; hypersensitive non-infectious rhinitis including combined (flower hypersensitivity) rhinitis, rhinitis with rhinorrhea, congestive rhinitis, edematous rhinitis and dry-nose type rhinitis; irritant-induced rhinitis including physical rhinitis, chemical rhinitis and radiation rhinitis; and atrophic rhinitis and idiopathic granulomatous rhinitis; sinusitis such as acute sinusitis, chronic sinusitis (maxillary empyema), eosinophilic sinusitis and paranasal cavity mycosis. The external preparation can be used for the purpose of treating or preventing one ore more of the above.

Combined rhinitis includes, for example, allergic rhinitis including perennial allergic rhinitis and seasonal allergic rhinitis, and nonallergic rhinitis including vasomotor (essential) rhinitis and eosinophilic rhinitis. Rhinitis with rhinorrhea includes, for example, gustatory rhinitis, cold air-induced rhinitis and senile rhinitis.

Congestive rhinitis includes, for example, drug-induced rhinitis, psychogenic rhinitis, gestational rhinitis, endocrine rhinitis and cold rhinitis.

Edematous rhinitis includes, for example, aspirin-hypersensitive rhinitis.

Among those mentioned above, it exerts an excellent effect specifically on hypersensitive non-infectious rhinitis, irritant-induced rhinitis and sinusitis, preferably on hypersensitive non-infectious rhinitis and chronic sinusitis, more preferably on combined (flower hypersensitivity) rhinitis, rhinitis with rhinorrhea, congestive rhinitis, edematous rhinitis and dry-nose type rhinitis, and chronic sinusitis. Therefore, the external preparation of the present invention can be used for the purpose of treating or preventing at least one or more rhinitis selected from the above-mentioned group.

In terms of its symptoms, for its effects as described above, it can be used for any of rhinitis with sneezing/rhinorrhea, rhinitis with nasal obstruction and rhinitis with both symptoms.

Immediately after a single application of 0.1 ml of 3 μg/ml to 500 μg/ml preparation was applied to the affected site in each nasal cavity, nasal discharge was stopped, nasal obstruction was improved, itches in nasal cavity mucosa or sneezing were stopped (immediate effect). A single application of a nasal drop induced persistent effects lasting for one day, or for a week or more in some cases (long-lasting effect). In addition, because it causes no local irritation at all, it can be used safely even in a subject who is not desired to use a nasal drop comprising rhinitis therapeutic that has conventionally been used such as steroid drug due to its strong local irritation.

Examples of multiple replaceable amino acids in the cyclic peptide of the invention are summarized below, based on working examples as described above, without not being limited thereto.

TABLE 32 Candidate multiple replacements of B ring Number Example of of Resi- Resi- Resi- Resi- replaced SEQ replace- due Amino due Amino due Amino due Amino amino acid ID ment ID acid ID acid ID acid ID acid sequence NO: 2 3 A, S 5 R CFSRRMDRISSSSGLGC 45 3 A, S 12 Q, T CFSRKMDRISSTSGLGC 46 3 A, S 13 T CFSRKMDRISSSTGLGC 47 3 A, S 15 M, I,  CFSRKMDRISSSSGIGC 48 V, A 3 A, S 16 A, S CFSRKMDRISSSSGLAC 49 5 R 12 Q, T CFGRRMDRISSTSGLGC 50 5 R 13 T CFGRRMDRISSSTGLGC 51 5 R 15 M, I, CFGRRMDRISSSSGIGC 52 V, A 5 R 16 A, S CFCRRMDRISSSSGLAC 53 12 Q, T 15 M, I, CFGRKMDRISSTSGIGC 54 V, A 12 Q, T 16 A, S CFGRKMDRISSTSGLAC 55 13 T 15 M, I, CFGRKMDRISSSTGIGC 56 V, A 13 T 16 A, S CFGRKMDRISSSTGLAC 57 15 M, I,  16 A, S CFGRKMDRISSSSGIAC 58 V, A 3 3 A, S 5 R 12 Q, T CFSRRMDRISSTSGLGC 59 3 A, S 5 R 13 T CFSRRMDRISSSTGLGC 60 3 A, S 5 R 15 M, I, V, A CFSRRMDRISSSSGIGC 61 3 A, S 5 R 16 A, S CFSRRMDRISSSSGLAC 62 3 A, S 12 Q, T 15 M, I, V, A CFSRKMDRISSTSG1GC 63 3 A, S 13 T 15 M, I, V, A CFSRKMDRISSSTGIGC 64 3 A, S 13 T 16 A, S CFSRKMDRISSSTGLAC 65 3 A, S 15 M, I, 16 A, S CFSRKMDRISSSSGLAC 66 V, A 5 R 12 Q, T 15 M, I, V, A CFGRRMDRISSTSGIGC 67 5 R 12 Q, T 16 A, S CFGRRMDRISSTSGIAC 68 5 R 13 T 15 M, I, V, A CFGRRMDRISSSTGIGC 69 5 R 13 T 16 A, S CFGRRMDRISSSTGLAC 70 5 R 15 M, I, 16 A, S CFGRRMDRISSSSGLAC 71 V, A 4 3 A, S 5 R 12 Q, T 15 M, I,  CFSRRMDR1SSTSGIGC 72 V, A 3 A, S 5 R 12 Q, T 16 A, S CFSRRMDRISSTSGLAC 73 3 A, S 5 R 13 T 15 M, I,  CFSRRMDRISSSTGIGC 74 V, A 3 A, S 5 R 13 T 16 A, S CFSRRMDRISSSTGLAC 75 3 A, S 5 R 15 M, I, V, A 16 A, S CFSRRMDRISSSSGIAC 76 

1.-15. (canceled)
 16. A method of using an external preparation comprising applying the external preparation to skin and/or mucosa of a subject, wherein the external preparation comprises a cyclic peptide having an amino acid sequence expressed by the Formula I:

wherein, X¹ denotes Gly, Val, Ala, Ser or Thr, X² denotes Arg, Gln or His, X³ denotes Lys or Arg, X⁴ denotes Met, Leu or Ile, X⁵ denotes Be or Val, X⁶ denotes Ser or Gly, X⁷ denotes Ser or Ala, X⁸ denotes Ser, Gln, Val, Ala, Thr, Leu, Ile or Met, X⁹ denotes Ser, Val, Ala or Thr, X¹⁰ denotes Gly or Arg, X¹¹ denotes Leu, Met, Ile, Val or Ala, X¹² denotes Gly, Ser or Ala, and the line connecting two Cys denotes a disulfide bond, and wherein said amino acid sequence does not have a peptide bond that is not between the amino acids constituting said amino acid sequence, or a derivative thereof or a pharmaceutically acceptable salt thereof.
 17. The method of using the external preparation according to claim 16, wherein the mucosa is labial, oral, nasal, ocular or vaginal mucosa.
 18. The method of using the external preparation according to claim 16 for treating and/or preventing dermatitis, for alleviating or resolving itch, for treating eczematous or other erosion or ulcer, or for skin-care.
 19. The method of using the external preparation according to claim 16 for treating and/or preventing alopecia, and/or for stimulating hair growth, and/or for growing hair.
 20. The method of using the external preparation according to claim 16 for treating and/or preventing rhinitis.
 21. (canceled)
 22. (canceled)
 23. The method according to claim 16, wherein X¹-X¹² are selected from the group consisting of following (1)-(12): (1) X¹ denotes Gly, (2) X² denotes Arg, Gln or His, (3) X³ denotes Lys or Arg, (4) X⁴ denotes Met, Leu or Ile, (5) X⁵ denotes Ile, (6) X⁶ denotes Ser or Gly, (7) X⁷ denotes Ser or Ala, (8) X⁸ denotes Ser, Gln, Val, Ala, Thr, Leu, Ile or Met, (9) X⁹ denotes Ser, Val, Ala or Thr, (10) X¹⁰ denotes Gly or Arg, (11) X¹¹ denotes Leu, and (12) X¹² denotes Gly, or a derivative thereof or a pharmaceutically acceptable salt thereof.
 24. The method according to claim 16 selected from the cyclic peptides of SEQ ID NOs: 3-8 and SEQ ID NOs: 16-75, or a derivative thereof or a pharmaceutically acceptable salt thereof.
 25. The method according to claim 16, wherein the derivative is substituted by a substituent which is capable of replacing a hydrogen atom, hydroxyl group, carboxy group, amino group or imino group in the cyclic peptide.
 26. The method according to claim 16, wherein the cyclic peptide is formed by deleting 1 to 4 amino acids in the cyclic peptide expressed by any one of the Formulae (I-a)-(I-e), or by replacing them with or adding them other amino acids, and wherein the cyclic peptide has an equal function with the cyclic peptide expressed by each of said formulae.
 27. The method according to claim 16, wherein the external preparation is an ingredient for a dermatitis therapeutic, dermatitis prophylactic, antipruritic, antiphlogistic, wound epithelialization-accelerating agent or skin-care product.
 28. The method according to claim 27, wherein the skin-care product is used for moisturizing, and/or for preventing or improving rough skin, and/or for sebum/acne care, and/or for alleviating irritation/anti-inflammation, and/or for skin-lightening, and/or for anti-aging, and/or for preventing/alleviating ultraviolet lesion, and/or for slimming, and/or for skin-cleansing.
 29. The method according to claim 16, wherein the external preparation is a bath agent, a body-cleansing agent or a hair-cleansing agent.
 30. The method according to claim 16, wherein the external preparation is an alopecia therapeutic, an alopecia prophylactic, a hair growing agent and/or a hair growth stimulant.
 31. The method according to claim 16, wherein the external preparation is a rhinitis therapeutic and/or rhinitis prophylactic.
 32. The method according to claim 16, wherein the external preparation is a cosmetic.
 33. The method according to claim 16, wherein the external preparation is a solid, semi-solid, powder, liquid, spray, ointment, cream, emulsion, gel or patch formulation.
 34. The method according to claim 16, wherein the external preparation is used as a pharmaceutical product, a quasi-drug or a cosmetic product. 